Secreted Insecticidal Protein and Gene Compositions From Bacillus Thuringiensis and Uses Therefor

ABSTRACT

The present invention relates to the isolation and characterization of nucleotide sequences encoding novel insecticidal proteins secreted into the extracellular space from  Bacillus thuringiensis  and related strains. The proteins are isolated from culture supernatants of  Bacillus thuringiensis  and related strains and display insecticidal activity against lepidopteran insects including European corn borer (ECB), tobacco budworm (TBW) and diamondback moth (DBM). Insecticidal proteins encoded by nucleotide sequences that hybridize under stringent conditions to the isolated and characterized nucleotide sequences are disclosed. Methods are disclosed for making and using transgenic cells and plants comprising the novel nucleotide sequence of the invention.

BACKGROUND OF INVENTION

The present invention relates to a new family of genes encodinglepidopteran-toxic proteins and insecticidal fragments thereof. Inparticular, the present invention is directed to exemplary proteinsdesignated herein as TIC900, TIC402, TIC403, TIC404, TIC961, TIC962,TIC963, TIC965 and TIC966, and insecticidal fragments thereof, eachencoded by exemplary nucleotide coding sequences designated hereinrespectively as tic900, tic402, tic403, tic404, tic434, tic961, tic962,tic963, tic965, and tic966, as well as to nucleotide sequence homologsthat (1) encode insecticidal proteins and (2) hybridize to the tic900,tic402, tic403, tic404, tic434, tic961, tic962, tic963, tic965, andtic966 coding sequences under stringent hybridization conditions. Thepresent invention also relates to host cells transformed with one ormore nucleotide sequences of the present invention or transformed withvariants of the nucleotide sequences set forth herein, genes related byidentity and/or similarity to the sequences set forth herein, and/orhomologs thereof, particularly those sequences that have been modifiedfor improved expression in plants. In a preferred embodiment, thetransformed host cells are plant cells.

Almost all field crops, plants, and commercial farming areas aresusceptible to attack by one or more insect pests. Particularlyproblematic are Coleopteran and Lepidoptern pests. For example,vegetable and cole crops such as artichokes, kohlrabi, arugula, leeks,asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets,bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon,crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni,carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas,parsnips, chicory, peas, chinese cabbage, peppers, collards, potatoes,cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga,eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach,green onions, squash, greens, sugar beets, sweet potatoes, turnip, swisschard, horseradish, tomatoes, kale, turnips, and a variety of spices aresensitive to infestation by one or more of the following insect pests:alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbagebudworm, cabbage looper, cabbage webworm, corn earworm, celeryleafeater, cross-striped cabbageworm, european corn borer, diamondbackmoth, green cloverworm, imported cabbageworm, melonworm, omnivorousleafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybeanlooper, tobacco budworm, tomato fruitworm, tomato hornworm, tomatopinworm, velvetbean caterpillar, and yellowstriped armyworm. Likewise,pasture and hay crops such as alfalfa, pasture grasses and silage areoften attacked by such pests as armyworm, beef armyworm, alfalfacaterpillar, European skipper, a variety of loopers and webworms, aswell as yellowstriped armyworms.

Fruit and vine crops such as apples, apricots, cherries, nectarines,peaches, pears, plums, prunes, quince almonds, chestnuts, filberts,pecans, pistachios, walnuts, citrus, blackberries, blueberries,boysenberries, cranberries, currants, loganberries, raspberries,strawberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate,pineapple, and tropical fruits are often susceptible to attack anddefoliation by achema sphinx moth, amorbia, armyworm, citrus cutworm,banana skipper, blackheaded fireworm, blueberry leafroller, cankerworm,cherry fruitworm, citrus cutworm, cranberry girdler, eastern tentcaterpillar, fall webworm, fall webworm, filbert leafroller, filbertwebworm, fruit tree leafroller, grape berry moth, grape leaffolder,grapeleaf skeletonizer, green fruitworm, gummosos-batrachedra commosae,gypsy moth, hickory shuckworm, hornworms, loopers, navel orangeworm,obliquebanded leafroller, omnivorous leafroller. omnivorous looper,orange tortrix, orangedog, oriental fruit moth, pandemis leafroller,peach twig borer, pecan nut casebearer, redbanded leafroller, redhumpedcaterpillar, roughskinned cutworm, saltmarsh caterpillar, spanworm, tentcaterpillar, thecla-thecla basillides, tobacco budworm, tortrix moth,tufted apple budmoth, variegated leafroller, walnut caterpillar, westerntent caterpillar, and yellowstriped armyworm.

Field crops such as canola/rape seed, evening primrose, meadow foam,corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice,safflower, small grains (barley, oats, rye, wheat, etc.), sorghum,soybeans, sunflowers, and tobacco are often targets for infestation byinsects including armyworm, asian and other corn borers, bandedsunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm(including southern and western varieties), cotton leaf perforator,diamondback moth, european corn borer, green cloverworm, headmoth,headworm, imported cabbageworm, loopers (including Anacamptodes spp.),obliquebanded leafroller, omnivorous leafier, podworm, podworm,saltmarsh caterpillar, southwestern corn borer, soybean looper, spottedcutworm, sunflower moth, tobacco budworm, tobacco hornworm, andvelvetbean caterpillar.

Bedding plants, flowers, ornamentals, vegetables and container stock arefrequently fed upon by a host of insect pests such as armyworm, azaleamoth, beet armyworm, diamondback moth, ello moth (hornworm), Floridafern caterpillar, Io moth, loopers, oleander moth, omnivorousleafroller, omnivorous looper, and tobacco budworm.

Forests, fruit, ornamental, and nut-bearing trees, as well as shrubs andother nursery stock are often susceptible to attack from diverse insectssuch as bagworm, blackheaded budworm, browntail moth, californiaoakworm, douglas fir tussock moth, elm spanworm, fall webworm, fruittreeleafroller, greenstriped mapleworm, gypsy moth, jack pine budworm,mimosa webworm, pine butterfly, redhumped caterpillar, saddlebackcaterpillar, saddle prominent caterpillar, spring and fall cankerworm,spruce budworm, tent caterpillar, tortrix, and western tussock moth.Likewise, pests such as armyworm, sod webworm, and tropical sod webwormoften attack turf grasses.

Because crops of commercial interest are often the target of insectattack, environmentally-sensitive methods for controlling or eradicatinginsect infestation are desirable in many instances. This is particularlytrue for farmers, nurserymen, growers, and commercial and residentialareas which seek to control insect populations using eco-friendlycompositions.

Bacillus thuringiensis is a gram-positive bacterium that producesproteinaceous crystalline inclusions during sporulation. These B.thuringiensis crystal proteins are often highly toxic to specificinsects. Insecticidal activities have been identified for crystalproteins from various B. thuringiensis strains against insect larvaefrom the insect orders Lepidoptera (caterpillars), Coleoptera (beetles)and Diptera (mosquitoes, flies).

Individual B. thuringiensis crystal proteins, also calleddelta-endotoxins or parasporal crystals or toxin proteins, can differextensively in their structures and insecticidal activities. Theseinsecticidal proteins are encoded by genes typically located on largeplasmids, greater than 30 mega Daltons (mDa) in size, that are found inB. thuringiensis strains. A number of these B. thuringiensis toxin geneshave been cloned and the insecticidal crystal protein productscharacterized for their specific insecticidal properties. Hofte et al.(1989) and Schnepf et al. (1998) provide reviews of B. thuringiensistoxin genes and crystal proteins.

The insecticidal properties of B. thuringiensis have been longrecognized, and B. thuringiensis strains have been incorporated incommercial biological insecticide products for over forty years.Commercial B. thuringiensis insecticide formulations typically containdried sporulated B. thuringiensis fermentation cultures whose crystalproteins are toxic to various insect species.

Traditional commercial B. thuringiensis bio-insecticide products arederived from “wild-type” B. thuringiensis strains, i.e., purifiedcultures of B. thuringiensis strains isolated from natural sources.Newer commercial B. thuringiensis bio-insecticide products are based ongenetically altered B. thuringiensis strains, such as the transconjugantB. thuringiensis strains described in U.S. Pat. Nos. 5,080,897 and4,935,353.

A characteristic of crystal proteins is their ability to coalesce toform crystals inside the B. thuringiensis mother cell. Upon lysis of themother cell the proteins are released as crystals into the externalenvironment. In addition, B. thuringiensis also produces non-crystalproteins that, in contrast to crystal proteins, are secreted by B.thuringiensis cells as soluble proteins into the culture medium.Secreted non-crystal proteins of B. thuringiensis includephospholipases, proteases, and β-lactamase that have little, if any,insecticidal activity. However, three secreted non-crystal proteins ofB. thuringiensis designated Vip1, Vip2 and Vip3 have been reported to betoxic to coleopteran or lepidopteran insects (Estruch et al., 1996; U.S.Pat. No. 5,866,326; WO94/21795; WO96/10083). A non-crystal protein of B.thuringiensis designated CryV is reported to be toxic to lepidopteraninsects (Kostichka et al., 1996). A large number of Bacillusthuringiensis isolates producing extracellular secreted insecticidaltoxin proteins have been identified by a number of differentinvestigators. Such isolates have all been shown to produce one or moreof these VIP or CryV toxin proteins or closely related homologs.Coleopteran inhibitory secreted BT proteins such as TIC901, TIC1201,TIC407, and TIC417 have been previously disclosed but appear to beunrelated to the proteins of the present invention (U.S. ProvisionalPatent Application No. 60/485,483 filed Jul. 7, 2003; PCT/US04/21692filed Jul. 6, 2004).

The inventors herein disclose a new class of extracellular secretedinsecticidal protein toxins that do not exhibit homology to the knownVIP or CryV classes of proteins. None of the one hundred thirty-sevenknown insect-toxic proteins of B. thuringiensis (Crickmore et al.,1998), more or less, are substantially related to the proteins of thepresent invention. In fact, no significant homology was found betweenthe sequences of the proteins of the present invention and any of thethousands of protein sequences contained in the National Center forGenome Resources (GenBank), Santa Fe, N. Mex.

SUMMARY OF INVENTION

In one embodiment, the present invention relates to an isolated andpurified insecticidal protein, exhibiting an amino acid sequencesubstantially as set forth in SEQ ID NO:4, (TIC900), SEQ ID NO:6(TIC402), SEQ ID NO:8 (TIC403), SEQ ID NO:10 (TIC404), SEQ ID NO:30(TIC434), SEQ ID NO:12 (TIC961), SEQ ID NO:14 (TIC962), SEQ ID NO:16(TIC963), SEQ ID NO:18 (TIC965), and SEQ ID NO:20 (TIC966), or relatedamino acid sequences and homologs thereof. Insecticidal activity ofTIC900 and related proteins have been demonstrated in bioassays withlepidopteran insects including European corn borer (ECB), tobaccobudworm (TBW) and Diamondback Moth (DBM), as shown herein.

In another embodiment, the present invention relates to an isolated andpurified nucleotide sequence, i.e. a coding sequence, comprising anucleotide sequence as set forth in SEQ ID NO:3 (tic900), SEQ ID NO:5(tic402), SEQ ID NO:7 (tic403), SEQ ID NO:9 (tic404), SEQ ID NO:29(tic434), SEQ ID NO:11 (tic961), SEQ ID NO:13 (tic962), SEQ ID NO:15(tic963), SEQ ID NO:17 (tic965), or SEQ ID NO: 19 (tic966), or relatedsequences or homologs thereof. The native tic900 coding sequence as setforth in SEQ ID NO:3 encodes the TIC900 protein exhibiting the aminoacid sequence as set forth in SEQ ID NO:4. Organisms producing TIC900 orrelated proteins exhibit insecticidal activity and/or insect-resistanceproperties. The native tic402 coding sequence as set forth in SEQ IDNO:5 encodes the TIC402 protein exhibiting the amino acid sequence asset forth in SEQ ID NO:6. The native tic403 coding sequence as set forthin SEQ ID NO:7 encodes the TIC403 protein exhibiting the amino acidsequence as set forth in SEQ ID NO:8. The native tic404 coding sequenceas set forth in SEQ ID NO:9 encodes the TIC404 protein exhibiting theamino acid sequence as set forth in SEQ ID NO:10. The native tic434coding sequence as set forth in SEQ ID NO:29 encodes the TIC434 proteinexhibiting the amino acid sequence as set forth in SEQ ID NO:30. Thenative tic961 coding sequence as set forth in SEQ ID NO:11 encodes theTIC961 protein exhibiting the amino acid sequence as set forth in SEQ IDNO:12. The native tic962 coding sequence as set forth in SEQ ID NO:13encodes the TIC962 protein exhibiting the amino acid sequence as setforth in SEQ ID NO:14. The native tic963 coding sequence as set forth inSEQ ID NO:15 encodes the TIC963 protein exhibiting the amino acidsequence as set forth in SEQ ID NO:16. The native tic965 coding sequenceas set forth in SEQ ID NO:17 encodes the TIC965 protein exhibiting theamino acid sequence as set forth in SEQ ID NO:18. The native tic966coding sequence as set forth in SEQ ID NO:19 encodes the TIC966 proteinexhibiting the amino acid sequence as set forth in SEQ ID NO:20. TIC900or related proteins and nucleotide sequences derived from Bt strainsthat encode these proteins are described herein as homologs of eachother, i.e., insecticidal proteins or insecticidal fragments thereofencoded by nucleotide sequences that hybridize to each or any of thesequences disclosed herein either under specific hybridizationconditions or under stringent hybridization conditions, and arespecifically intended to be included within the scope of the presentinvention.

In a further embodiment, the present invention relates to a biologicallypure culture of a Bacillus thuringiensis bacterium transformed with aplasmid vector containing a nucleotide sequence as set forth in SEQ IDNO:3 (tic900), SEQ ID NO:5 (tic402), SEQ ID NO:7 (tic403), SEQ ID NO:9(tic404), SEQ ID NO:29 (tic434), SEQ ID NO:11 (tic961), SEQ ID NO:13(tic962), SEQ ID NO:15 (tic963), SEQ ID NO:17 (tic965), or SEQ ID NO: 19(tic966), or a related sequence or homolog that produces an insecticidalprotein and secretes the protein into the extracellular spacesurrounding the bacterial strain during fermentation. An exemplarystrain SIC9002 has been deposited in the Northern Regional ResearchLaboratory of Agricultural Research Service Center Collection (NRRL),USDA, 1815 North University Street, Peoria, Ill. 61604, pursuant to theBudapest Treaty on the International Recognition of the Deposit ofMicroorganism for the Purposes of Patent Procedure on Apr. 25, 2000 andhas been assigned the accession No. NRRL B-30582. One plasmid containingthe tic900 nucleotide sequence is set forth herein as pBD1.

In a further embodiment, the invention also relates to a biologicallypure culture of a B. thuringiensis bacterium designated as strain EG5438exhibiting insecticidal activity against lepidopteran insects. B.thuringiensis strain EG5438 represents a wild type B. thuringiensisstrain from which a tic900 coding sequence was isolated. The strain hasbeen deposited in the NRRL, USDA, pursuant to the Budapest Treaty on May3, 2000 and has been assigned the accession No. NRRL B-30584.

In a further embodiment, the present invention provides a nucleotidesequence as set forth in SEQ ID NO:3 encoding a TIC900 amino acidsequence (SEQ ID NO:4), and an oligonucleotide portion that can belabeled and used as a hybridization probe for identifying additionalrelated genes encoding related insecticidal proteins or homologuesthereof. Other related nucleotide sequences specifically exemplifiedherein comprise sequences as set forth in SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, and SEQ ID NO:19, each of which encode insecticidal proteintoxins as set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:30, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQID NO:20, respectively.

In yet a further embodiment, the invention provides plant cells andplants that have been transformed with a nucleotide sequence encoding aTIC900 or related protein as set forth in SEQ ID NO:4 or insecticidalfragment thereof, or a TIC900 protein homolog thereof, selected from thegroup consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:30, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQID NO:20. The nucleotide sequence can be translated and expressed byplant cells and in plant tissues at levels sufficient to inhibit or killlepidopteran insect pests that come into contact with the transgenicplant expressing said protein, particularly when said pests ingest partsof said transgenic plant. Both monocot and dicot plants are within thescope of the invention. Modification of the sequence may be required inorder to affect the maximum level of expression and to enhance theability of the plant containing the sequence to produce insecticidallevels of the TIC900 or related protein. Transformation of plants withthe nucleotide sequences disclosed herein may result in increasedfrequency of transformants that express the transgene, i.e., tic900 orits homolog, as well as the generation of a greater percentage oftransformation events exhibiting morphologically normal physiology.

In yet a further embodiment, the present invention also provides amethod for producing a transgenic plant that exhibits increasedexpression levels of a nucleotide sequence encoding a TIC900 protein orinsecticidal fragment thereof or its homolog and thereafter increasedlevels of the insecticidal TIC900 protein or its homolog. Thus theplants transformed with the nucleotide sequences disclosed hereinexhibit improved and increased levels of lepidopteran pest resistanceabilities in comparison to a plant lacking a nucleotide sequenceencoding a TIC900, an insecticidal fragment of a TIC900, or one of itshomologs.

In accomplishing the foregoing, a method for expressing a nucleotidesequence encoding a TIC900 protein or its homolog in a plant is providedcomprising the steps of a) inserting into the genome of a plant cell anucleic acid sequence comprising in the 5′ to 3′ direction, a plantfunctional promoter operably linked to a structural DNA sequenceoptimized for plant expression that causes production of an RNA sequenceencoding all of or an insecticidal fragment of a TIC900 polypeptidesequence as set forth in SEQ ID NO:4, or its homolog selected from thegroup consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:30, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQID NO:20, or a sequence having at least from about 80%, or from at leastabout 85%, or from at least about 90%, or from at least about 95%, orfrom at least about 99% sequence identity to the amino acid sequence asset forth in SEQ ID NO:4, or a sequence encoding an insecticidal proteinthat hybridizes to any of these sequences under either specific orstringent hybridization conditions, and a 3′ non-translated DNA sequencethat functions in the cells of the plant to cause transcriptiontermination and polyadenylation; b) obtaining transformed plant cellscontaining the nucleic acid sequence; and c) generating from thetransformed plant cells genetically transformed plants that express thenucleotide sequence encoding the TIC900 or a related protein, whereinthe transformed plants are morphologically normal and exhibit elevatedor improved levels of lepidopteran pest resistance compared to a plantnot transformed to express said protein.

Another embodiment of the present invention is the provision forantibodies that bind specifically to epitopes presented only by theTIC900 protein or its homologs. Antibodies can be used for identifyingthe presence of a TIC900 protein or a homolog, for purifying the proteinor homolog, for identifying a nucleotide sequence from which a TIC900protein or a homolog is being expressed, and for use in kits designed toallow the detection of a TIC900 protein or a homolog or the detection ofa nucleotide sequence expressing the protein or homolog.

The inventors contemplate that the protein compositions disclosed hereinwill find particular utility as insecticides for topical and/or systemicapplication to field crops, grasses, fruits and vegetables, andornamental plants. In a preferred embodiment, the bioinsecticidecomposition comprises an oil flowable suspension of bacterial cellswhich expresses a novel insecticidal protein disclosed herein.Preferably the cells are B. thuringiensis EG5438 or SIC9002 cells,however, any such bacterial host cell expressing the novel nucleic acidsegments disclosed herein and producing a crystal protein iscontemplated to be useful, such as B. megaterium, B. subtilis, E. coli,or Pseudomonas spp.

A particular advantage of the present invention comprises an improvementin insect resistance management (IRM). The ability to combine two ormore insecticidal agents, each toxic to the same insect pest species,into a single composition, and each agent exhibiting a mode of actiondifferent from the other insecticidal agents with which it is combined,present a means for more effectively controlling a particular insectpest species by substantially reducing the likelihood that resistance tothe insecticidal composition will develop in a population. The TIC900protein an insecticidal fragment thereof, or any homolog thereof, of thepresent invention can be combined with any number of known insecticidalagents to achieve the level of resistance management in a particularcomposition, preferably by expression of the combination of insecticidalagents in plants. In particular TIC900 or related insecticidal proteincompositions can be combined with a Cry1 or Cry2 amino acid sequence ora variant thereof to achieve control of various lepidopteran plant pestspecies, or with other appropriate Cry proteins, and with variousinsecticidal compositions derived from Xenorhabdus and Photorhabdusbacterium species that have been shown to exhibit insecticidalbioactivity directed to lepidopteran plant pest species. Preferably thein planta use of these compositions would be directed to enhancedexpression of the proteins in the parts of the plant that exhibit thegreatest vulnerability to lepidopteran insect predation. For protectionof maize species against European corn borer (ECB), it would bepreferable to achieve the highest levels of expression in the leaves andstems of the plant. For tobacco species susceptible to budworm, it wouldbe preferable to achieve the highest levels of expression in thesprouting parts of the plant, i.e., within the bud systems of the plant.For protection of a cruciferous vegetable species against diamondbackmoth (DBM), it would be preferable to achieve the highest levels ofexpression in the leaves and stems of the plant.

The insecticidal proteins of the present invention can also be combinedwith insecticidal and/or fungicidal toxins expressed in planta toachieve a recombinant plant that exhibits multiple levels of resistanceto infestation by pests that are not beneficial to plants. For example,a protein of the present invention can be expressed along with a proteinthat exhibits coleopteran insect control, and/or along with a protein orother agent that exhibits antifungal activity, to achieve a recombinanttransgenic plant that exhibits improved resistance to lepidopteraninsect pests, coleopteran insect pests, and fungal pests. Otherpermutations of levels of resistance are known to those of skill in theart, such as means for resistance to piercing and sucking insectinfestation, and nematode infestation, etc. The insecticidal proteins ofthe present invention can also be combined with one or more nucleotidesequences expressed as one or more dsRNA's for use in suppression of oneor more genes (1) in the target pest as a means for achieving a plantthat exhibits multiple layers of resistance to infestation by aparticular pest, (2) in the plant as a means for achieving desired planttraits, or (3) in various combinations to achieve the desired propertiesof (1) or (2) collectively.

Chimeric proteins consisting of all or a part of one or more proteins ofthe present invention fused to other proteins that are useful in plantprotection from infestation or otherwise are contemplated herein. Forexample, domains of the proteins of the present invention have beenfound to exhibit a low level of similarity to other Bt toxins, such asCry3Aa toxin domain I, Cry1Ca toxin domain II, and Cry1Ja toxin domainIII (in particular, Domains I, II, and III of the toxin portion of theTIC900 protein, respectively). The proteins of the present invention canbe fused to the protoxin domains of any of the Cry1 proteins known inthe art, resulting in crystal toxin protein formation when expressed inBt or other Bacillus strains of bacteria. Furthermore, the domainsidentified herein within the amino acid sequence of the proteins of thepresent invention can be exchanged with other similar domains frominsecticidal Bt toxin proteins to achieve improved insecticidal activityand/or host ranges that have not previously been observed with Cry1toxin domain exchanges (Malvar et al. U.S. Pat. No. 6,017,534; Galizziet al, PCT/EP90/0114, WO 91/01087).

Another embodiment comprises an isolated polynucleotide that encodes aBacillus thuringiensis insecticidal toxin or insecticidal fragmentthereof, active against an insect pest, wherein the toxin orinsecticidal fragment has a molecular weight between approximately65,000 Daltons and approximately 70,000 Daltons. In addition, thenucleotide sequence encoding the toxin, or the complement thereof,hybridizes under specific or stringent hybridization conditions to SEQID NO:3. The toxin preferably exhibits biological activity incontrolling or killing a lepidopteran insect pest, preferably Europeancorn borer (ECB), tobacco budworm (TBW) and/or diamondback moth (DBM).In one embodiment the nucleotide sequence encoding the toxin isoptimized for expression in plants, yet encodes substantially the toxinor an insecticidal fragment thereof, i.e., encodes the same orsubstantially the same amino acid sequence as present in the nativeamino acid sequence.

Another embodiment of the present invention provides for host cellstransformed to contain a polynucleotide encoding an insecticidal proteinof the present invention or an insecticidal fragment thereof. Preferablythe nucleotide sequences of the present invention are modified toimprove expression of the proteins of the present invention in apreferred host cell. The host cell of the present invention is selectedfrom the group consisting of a bacterial cell, a fungal cell, and aplant cell. Expression in a plant cell can comprise expression toachieve accumulation of the insecticidal protein in the cytoplasm, orcan result in the insecticidal protein being accumulated into asubcellular organelle such as a plastid, chloroplast, or mitochondria.Alternatively the insecticidal protein of the present invention orinsecticidal fragments thereof could be localized to the proteinsecretion machinery of the particular host cell and result in anaccumulation of the protein product outside of the cell and into theextracellular spaces surrounding the cell.

An additional embodiment of the present invention provides a method forcontrolling infestation of a plant by a lepidopteran insect species.Preferably a pesticidal amount of an insecticidal protein of the presentinvention or insecticidal fragment thereof is provided for consumptionby the insect pest in the diet of the insect. The diet can consist of aplant part that the insect normally feeds upon, such as a plant tissueor plant cell. The insecticidal protein or insecticidal fragment thereofcan be provided in a composition that is applied to the surface of theplant tissue, plant part, or plant cell or more preferably can beproduced by the protein synthesis machinery of the cell and, asdescribed above, accumulated within the plant cell or secreted outsideof the plant cell, so long as the amount of the protein toxin providedis an insecticidal amount sufficient to inhibit the insect pest fromfurther feeding, or to inhibit the further growth and development of theinsect pest, or to cause mortality to the insect pest. The insecticidaltoxin or fragment thereof is derived from a nucleotide sequence that isencoded in Bacillus thuringiensis by a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequencesubstantially complementary to SEQ ID NO:3.

The present invention also provides a method for detecting a firstnucleotide sequence that hybridizes to a second nucleotide sequence asset forth in SEQ ID NO:3, wherein the first nucleotide sequence encodesan insecticidal protein or insecticidal fragment thereof and hybridizesunder specific or stringent hybridization conditions to the secondnucleotide sequence. Other exemplary second nucleotide sequences are SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19.

It is also contemplated that the proteins of the present invention wouldbe useful when expressed in planta to provide an improved level ofprotection from insect infestation to plants expressing the proteins orinsecticidal fragments thereof. Therefore it is envisioned that one ormore nucleotide sequences encoding a TIC900 insecticidal protein orinsecticidal fragment thereof or homolog thereof, or combinationsthereof, whether expressed individually or as chimeras or as fusions,would be introduced into the plant cell, either into the genome, intothe chloroplast or mitochondrial DNA, or into an organelle as a stableand autonomously replicating extra-chromosomal element, for expressionof the said TIC900 protein or insecticidal fragment thereof or homologthereof. Preferably the sequence is a non-naturally occurring nucleotidesequence that encodes the insecticidal protein or insecticidal fragmentthereof. Plant cells transformed with such sequences are provided forherein. Plants grown from the transformed plant cells are provided bythe instant inventions. Seeds and progeny of the seeds from thetransformed plants of the present invention are also provided so long asthe seeds contain at least the sequences encoding the insecticidalproteins or insecticidal protein fragments thereof. The nucleotidesequences envisioned are at least from about 60 to about 85% identicalto the nucleotide sequences of the present invention as isolated from B.thuringiensis.

Exemplary sequences of the present invention include at least, inaddition to those related to SEQ ID NO:5 and SEQ ID NO:4: (1) thenucleotide sequence as set forth in SEQ ID NO:5, and the amino acidsequence encoded by SEQ ID NO:5 as set forth in SEQ ID NO:6, alsoreferred to herein as insecticidal protein TIC402; (2) the nucleotidesequence as set forth in SEQ ID NO:7, and the amino acid sequenceencoded by SEQ ID NO:7 as set forth in SEQ ID NO:8, also referred toherein as insecticidal protein TIC403; (3) the nucleotide sequence asset forth in SEQ ID NO:9, and the amino acid sequence encoded by SEQ IDNO:9 as set forth in SEQ ID NO:10, also referred to herein asinsecticidal protein TIC404; (4) the nucleotide sequence as set forth inSEQ ID NO:29, and the amino acid sequence encoded by SEQ ID NO:29 as setforth in SEQ ID NO:30, also referred to herein as insecticidal proteinTIC434; (5) the nucleotide sequence as set forth in SEQ ID NO:11, andthe amino acid sequence encoded by SEQ ID NO:11 as set forth in SEQ IDNO:12, also referred to herein as insecticidal protein TIC961; (6) thenucleotide sequence as set forth in SEQ ID NO:13, and the amino acidsequence encoded by SEQ ID NO:13 as set forth in SEQ ID NO:14, alsoreferred to herein as insecticidal protein TIC962; (7) the nucleotidesequence as set forth in SEQ ID NO:15, and the amino acid sequenceencoded by SEQ ID NO:15 as set forth in SEQ ID NO:16, also referred toherein as insecticidal protein TIC963; (8) the nucleotide sequence asset forth in SEQ ID NO:17, and the amino acid sequence encoded by SEQ IDNO:17 as set forth in SEQ ID NO:18, also referred to herein asinsecticidal protein TIC965; and (9) the nucleotide sequence as setforth in SEQ ID NO:19, and the amino acid sequence encoded by SEQ IDNO:19 as set forth in SEQ ID NO:20, also referred to herein asinsecticidal protein TIC966. Each of these proteins and the native B.t.nucleotide sequences encoding these proteins are related to TIC900 asdefined herein. For example, and respectively, SEQ ID NO:5 is anucleotide sequence encoding a TIC402 insecticidal protein as set forthin SEQ ID NO:6. SEQ ID NO:5 as shown herein is identifiable byhybridization to SEQ ID NO:3 under stringent conditions. SEQ ID NO:5encodes a protein that exhibits lepidopteran toxic biological activity,exhibiting toxicity to European corn borer (ECB), tobacco budworm (TBW)and/or diamondback moth (DBM). SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19are each capable of hybridizing to each other under stringentconditions, and each sequence can be identified by hybridization to SEQID NO:3 under stringent conditions, and each sequence can be identifiedby amplification using the oligonucleotide primers as set forth in SEQID NO:21 and SEQ ID NO:22. The primers as set forth in SEQ ID NO:21 andSEQ ID NO:22 are diagnostic for identifying the presence of a nucleotidesequence encoding a TIC900 or related insecticidal protein in a sample.These oligonucleotides, when used together under defined amplificationconditions and in the presence of a suitable nucleotide sequencesubstrate, produce an amplicon that is diagnostic for the presence of aTIC900 coding sequence or a homolog thereof. This particular reaction isuseful for detecting the presence of a B.t. gene encoding aninsecticidal protein corresponding to a TIC900 or related protein in asample, and greatly simplifies the search for and identification of suchrelated sequences.

Kits for detecting the presence of the nucleotide sequences of thepresent invention are also contemplated. Such kits contain one or morenucleotide sequences each for use as a probe for detecting the presenceof a nucleotide sequence encoding an insecticidal protein of the presentinvention or fragment thereof. Such kits could also or alternativelycontain antibody specific for binding to one or more peptides of theproteins of the present invention, as well as reagents for use with theprobe or antibody, and the kits would also contain control samples foruse in ensuring that the nucleotides or peptides identified with theprobe and or antibody and reagents were functioning according to themanufacturers” instructions. All of the reagents necessary for carryingout the methods of identification of either nucleotide sequences orpeptides would be packaged together in a kit along with instructions foruse. An exemplary kit could contain a TIC900 or related nucleotidesequence encoding an insecticidal protein along with a sample of theexemplary nucleotide sequence amplification primers as set forth in SEQID NO:21 and SEQ ID NO:22, together with the necessary reagentsnecessary for carrying out an amplification reaction, all packagedtogether in the kit.

A plant or plant tissue transformed to contain (a) a nucleotide sequenceencoding one or more of the proteins of the present invention, (2) allor an insecticidally active portion of one or more of the proteins ofthe present invention, or (3) a chimera containing all or any portion ofone or more proteins of the present invention can be detected using anynumber of means well known in the art including but not limited tonucleotide sequence based detection methods and/or protein baseddetection methods. Agronomically and commercially important productsand/or compositions of matter derived from such transformed plants orplant tissues include but are not limited to animal feed, commodities,and corn, soy, cotton, canola, wheat, oat, rice, sugar-cane, chick-pea,and cow-pea products and by-products that are intended for use as foodfor human consumption or for use in compositions that are intended forhuman consumption including but not limited to flours, meals, syrups,oil, starch, popcorn, cakes, cereals containing the fruits and seeds ofthese crops and by-products, and the like are intended to be within thescope of the present invention if these products and compositions ofmatter contain detectable amounts of the nucleotide sequences encodingthe proteins or derivatives of the proteins as set forth herein.

Plants or plant parts suspected of containing a protein or nucleotideencoding a protein of the present invention in a biological sample canbe detected using the method comprising the steps of contacting a samplesuspected of containing said nucleotide with a polynucleotide probe thathybridizes under stringent hybridization conditions with said nucleotideand that does not hybridize under stringent hybridization conditionswith a nucleotide from a control plant, subjecting said sample and saidprobe to said stringent hybridization conditions, and detecting thehybridization of said probe to the nucleotide.

One embodiment of the present invention comprises a biological samplederived from a transgenic plant, tissue, or seed, wherein the samplecomprises a nucleotide sequence which is or is complementary to asequence encoding a protein of the present invention, and wherein saidsequence is detectable in said sample using a nucleic acid amplificationor nucleic acid hybridization method. The sample can consist of a samplethat is selected from the group consisting of an extract obtainable fromthe transgenic plant containing the nucleotide sequence, and the extractcan contain any nucleotide sequence encoding one or more of the proteinsof the present invention, or the complement thereof. The biologicalsample is preferably selected from the group consisting of a flour suchas corn flour, a meal such as corn meal, a syrup such as corn syrup, anoil such as corn oil, cotton oil, linseed oil, soybean or canola oil,safflower oil, sunflower oil, peanut oil, and the like, a starch such ascorn starch, and any cereal that can be manufactured in whole or in partto contain grain or grain by-products. The nucleotide sequence isdetectable in the extract using a nucleic acid amplification or nucleicacid hybridization method.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 represents an amino acid sequence deduced by Edmunddegradation of a 14 kDa cyanogen bromide fragment of a TIC900 proteinand corresponds to amino acid positions 397-414 as set forth in SEQ IDNO:4.

SEQ ID NO:2 represents the nucleotide sequence of a hybridization probedesignated as WD470 designed based upon the amino acid sequence as setforth in SEQ ID NO:1, for use in detecting nucleotide sequences encodingTIC900 and related proteins.

SEQ ID NO:3 represents a native Bacillus thuringiensis nucleotidesequence consisting of 1803 consecutive nucleotides encoding a TIC900insecticidal protein consisting of 601 amino acid as set forth in SEQ IDNO:4.

SEQ ID NO:4 represents the TIC900 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:3.

SEQ ID NO:5 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC402.

SEQ ID NO:6 represents the TIC402 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:5.

SEQ ID NO:7 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC403.

SEQ ID NO:8 represents the TIC403 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:7.

SEQ ID NO:9 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC404.

SEQ ID NO:10 represents the TIC404 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:9.

SEQ ID NO:11 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC961.

SEQ ID NO:12 represents the TIC961 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:11.

SEQ ID NO:13 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC962.

SEQ ID NO:14 represents the TIC962 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:13.

SEQ ID NO:15 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC963.

SEQ ID NO:16 represents the TIC963 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:15.

SEQ ID NO:17 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC965.

SEQ ID NO:18 represents the TIC965 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:17.

SEQ ID NO:19 represents a tic900 homologous nucleotide sequence encodinga native Bacillus thuringiensis TIC900 related protein, designatedherein as TIC966.

SEQ ID NO:20 represents the TIC966 amino acid sequence deduced from thenucleotide sequence as set forth in SEQ ID NO:19.

SEQ ID NO:21 represents a 5′ end sequence primer used as a probe thatbinds specifically to TIC900 homologous sequences.

SEQ ID NO:22 represents a 3′ end sequence primer used as a probe thatbinds specifically to TIC900 homologous sequences.

SEQ ID NO:23 represents a tic109 nucleotide sequence encoding a TIC109chimeric protein consisting of a nucleotide sequence encoding a TIC900insecticidal protein domain linked in frame to a nucleotide sequenceencoding a Cry1Ac protoxin domain fragment.

SEQ ID NO:24 represents a TIC109 chimeric protein amino acid sequenceconsisting of a TIC900 insecticidal amino acid sequence (1-603) linkedto a Cry1Ac protoxin domain fragment amino acid sequence (606-1168).

SEQ ID NO:25 represents a tic110 nucleotide sequence encoding a TIC110chimeric protein consisting of a nucleotide sequence encoding a Cry1Ftoxin domain I fragment (nucleotides 1-723) linked in frame to anucleotide sequence encoding a TIC900 toxin fragment domain II-III(nucleotides 724-1809) linked in frame to a nucleotide sequence encodinga Cry1Ac protoxin domain fragment (nucleotides 1810-3510).

SEQ ID NO:26 represents a TIC110 chimeric protein amino acid sequenceconsisting of a Cry1F toxin domain I fragment (amino acids 1-233) linkedto a TIC900 toxin domain II-III fragment (amino acids 234-603) linked toa Cry1Ac protoxin domain fragment (amino acids 604-1170).

SEQ ID NO:27 represents a tic111 nucleotide sequence encoding a TIC111chimeric protein consisting of a nucleotide sequence encoding a Cry1Actoxin domain I fragment (nucleotides 1-705) linked in frame to anucleotide sequence encoding a TIC900 toxin domain II-III fragment(nucleotides 706-1815) linked in frame to a nucleotide sequence encodinga Cry1Ac protoxin domain fragment (nucleotides 1822-3516).

SEQ ID NO:28 represents a TIC111 chimeric protein amino acid sequenceconsisting of a Cry1Ac toxin domain I fragment (amino acids 1-235)linked to a TIC900 toxin domain II-III fragment (amino acids 236-605)linked to a Cry1Ac protoxin domain fragment (amino acids 608-1172).

SEQ ID NO:29 represents a B. thuringiensis strain EG4611 about 7.5 kbnucleotide sequence containing a TIC434 coding sequence, said codingsequence being from about nucleotide position 425 through aboutnucleotide position 2238.

SEQ ID NO:30 represents a TIC434 amino acid sequence.

SEQ ID NO:31 represents a chimeric sequence encoding a TIC435 amino acidsequence corresponding to a TIC434 amino acid sequence fused in frame toa sequence encoding a Cry1 protoxin amino acid sequence; said TIC434amino acid sequence coding region corresponding to about nucleotideposition 1 through about nucleotide position 1825, and said Cry1protoxin amino acid sequence coding region corresponding to aboutnucleotide position 1826 through about nucleotide position 3525.

SEQ ID NO:32 represents a chimeric TIC435 amino acid sequence.

DETAILED DESCRIPTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the detailed description should not be construed to unduly limit thepresent invention as modifications and variations in the embodimentsdiscussed herein may be made by those of ordinary skill in the artwithout departing from the spirit or scope of the present inventivediscovery.

In accordance with the present invention, a new genus of nucleotidesequences encoding insecticidal proteins derived from Bacillusthuringiensis and related Bacillus strains has been discovered. Asdefined elsewhere herein, these nucleotide sequences all hybridize toeach other under stringent conditions. The proteins encoded by thesenucleotide sequences each exhibit lepidopteran species inhibitorybiological activity, and so are considered to be insecticidal proteins.Each of the proteins encoded by these nucleotide sequences can beexpressed in plants alone or in combinations with each other or withother lepidopteran inhibitory insecticidal agents such as proteins,crystal proteins, toxins, and/or pest specific double stranded RNA'sdesigned to suppress genes within one or more target pests, and the liketo achieve a means of insect resistance management in the field that hasnot feasible before by merely using the known lepidopteran insecticidalproteins derived from Bacillus thuringiensis strains, such as Cry1proteins and various lepidopteran inhibitory insecticidal proteinsderived from Bacillus laterosporous species and Bacillus sphaericusspecies. The proteins of the present invention can also be used inplants in combination with other types of insecticidal toxins forachieving plants transformed to contain at least one means forcontrolling one or more of each of the common plant pests selected fromthe groups consisting of lepidopteran insect pests, coleopteran insectpests, piercing and sucking insect pests, and the like. The proteins ofthe present invention are also contemplated for use in formulations,either alone or in combinations with other insecticidal agents, asinsecticides for topical and/or systemic application to field crops,grasses, fruits and vegetables, and ornamental plants. In a preferredembodiment, the bio-insecticide composition comprises an oil flowablesuspension of bacterial cells that expresses one or more of a novelinsecticidal protein disclosed herein. Preferably the cells are B.thuringiensis EG5438 or SIC9002 cells, however, any such bacterial hostcell expressing the novel nucleic acid segments disclosed herein andproducing a crystal protein

The insecticidal proteins of the present invention may also be used incompositions for controlling insect infestation of plants either aloneor in combination with other insecticidal proteins or agents, and mayalso be used alone or in combination with gene suppressionmethodologies. As used herein “gene suppression” means any of thewell-known methods for suppressing expression of protein from a geneincluding post transcriptional gene suppression and transcriptionalsuppression.

As used herein an “pest resistance” trait is a characteristic of atransgenic plant is resistant to attack from a plant pest such as avirus, a nematode, a larval insect or an adult insect that typically iscapable of inflicting crop yield loss in a progenitor plant. Such pestresistance can arise from a natural mutation or more typically fromincorporation of recombinant DNA that confers pest resistance. To impartinsect resistance to a transgenic plant such recombinant DNA can, forexample, encode an insect lethal protein such as a delta endotoxin ofBacillus thuringiensis bacteria, e.g. as is used in commerciallyavailable varieties of cotton and corn, encode an insecticidal toxinprotein disclosed herein such as a TIC900 or related protein orinsecticidal fragment thereof, or be transcribed to a double-strandedRNA targeted for suppression of an essential gene in the insect, or anycombination of these insecticidal agents. To illustrate that theproduction of transgenic plants with pest resistance is a capability ofthose of ordinary skill in the art reference is made to U.S. Pat. Nos.5,250,515; 5,880,275 and 6,555,655 which disclose plants expressing anendotoxin of Bacillus thuringiensis bacteria. See also U.S. Pat. No.6,506,599 (Fire et al.) and U.S. Patent Application Publication2003/0061626 A1 (Plaetinck et al.) and U.S. Patent ApplicationPublication 2003/0150017 A1 (Mesa et al.) which disclose control ofinvertebrates by permitting the pest to feed on transgenic plants whichproduce double-stranded RNA for suppressing a target gene in the pest.See also U.S. Pat. No. 5,986,175 (Jilka et al.) that discloses thecontrol of viral pests by transgenic plants which express viralreplicase. All of the above-described patents and applicationsdisclosing materials and methods for pest control in plants areincorporated herein by reference.

Surprisingly, the proteins of the present invention appear to beunrelated to any of the Bacillus thuringiensis insecticidal proteinsheretofore discovered in the art. The proteins of the present inventionare shown herein to be excreted into the extracellular space surroundingthe Bacillus species from which they are derived. These proteins areshown herein to be significantly smaller than the previously known Cryproteins in the art, and are expressed during the vegetative stage ofgrowth of the isolated and purified bacterial cell cultures. This isunlike the expression of Cry proteins which are expressed generally inthe sporulation phase of growth and which form various crystallinebodies within the forespore of the cell.

As will become apparent to those of skill in the art, the inventorsherein disclose the isolation and purification of a nucleotide sequence,tic900, encoding a precursor TIC900 protein (TIC900p) that issubsequently processed to release a mature TIC900 protein (TIC900m) thatexhibits lepidopteran species inhibitory biological activity. Theinventors herein disclose the use of the tic900 sequence as a means foridentifying a multitude of other homologs and related sequences, whicheach also encode insecticidal proteins related to TIC900.

Nucleotide sequences disclosed herein and encoding TIC900 and relatedproteins were derived from various strains of Bacillus thuringiensis,i.e., the strain EG5438 contained at least one gene designated herein astic900. The strain EG5438 was deposited under the provisions of theBudapest Treaty with the permanent collection of the NRRL on May 3, 2002and was provided with the NRRL accession No. NRRL B-30584. Anotherstrain identified herein to contain a sequence encoding TIC900, anucleotide sequence identical to the EG5438 tic900 allele, was B.thuringiensis strain EG5526.

Nucleotide sequences related to tic900, and amino acid sequences relatedto TIC900 (including precursor and mature species of TIC900) which aredisclosed herein include but are not limited to tic402 and the encodedinsecticidal protein TIC402 isolated from and produced at least by B.t.strains EG3879, tic403 and the encoded insecticidal protein TIC403isolated from and produced at least by B.t. strain EG4332, tic404 andthe encoded insecticidal protein TIC404 isolated from and produced atleast by B.t. strain EG4971, tic434 and the encoded insecticidal proteinTIC434 isolated from and produced at least by B.t. strain EG4611, tic961and the encoded insecticidal protein TIC961 isolated from and producedat least by B.t. strain EG4090, tic962 and the encoded insecticidalprotein TIC962 isolated from and produced at least by B.t. strainEG4293, tic963 and the encoded insecticidal protein TIC963 isolated fromand produced at least by B.t. strain EG4611, tic965 and the encodedinsecticidal protein TIC965 isolated from and produced at least by B.t.strain EG5023, and tic966 and the encoded insecticidal protein TIC966isolated from and produced at least by B.t. strain EG4092.

It is intended that the proteins of the present invention be used foragricultural purposes, i.e., for protecting plants from insect pestinfestation, and more particularly for protecting plants fromlepidopteran insect pest infestation. As exemplified herein, theproteins of the present invention are useful for protecting plants atleast from European corn borer (ECB) infestation, at least from tobaccobudworm (TBW) infestation and at least from diamondback moth (DBM)infestation. Plant protection can be achieved by topical application ofa plant or plant parts such as by applying to the surface of the plant,i.e., the leaves, flowers, stems, stalks, and roots, a composition thatcontains an insecticidally effective amount of one or more of theproteins of the present invention. Alternatively, and preferably, theplant itself will be transformed to contain a nucleotide sequencemodified for improved expression of the protein of the present inventionin planta or expression of an insecticidal portion thereof.

The TIC900 protein is an insecticidal compound active againstlepidopteran insects such as ECB, TBW and DBM. The TIC900 protein as setforth in SEQ ID NO:4 and related insecticidal proteins may be used asthe active ingredient in insecticidal formulations useful forcontrolling lepidopteran insects. As used herein and with reference toinsecticidal proteins that are related to TIC900, it is intended thatrelated insecticidal proteins are those that are identified as homologsof TIC900 or those that are identified as being encoded by a nucleotidesequence that hybridizes under stringent conditions to all or a part ofthe native Bacillus thuringiensis sequence encoding the TIC900 proteinor an insecticidal portion thereof. Of course, one skilled in the artwill recognize that, due to the redundancy of the genetic code, manyother sequences are capable of encoding such related proteins, and thosesequences, to the extent that they function to express insecticidalproteins either in Bacillus strains or in plant cells, are intended tobe encompassed by the present invention, recognizing of course that manysuch redundant coding sequences will not hybridize under stringentconditions to the native sequence encoding TIC900. Coding sequences areconceivable that function to encode all or an insecticidal portion of aTIC900 or related protein that do not hybridize under stringentconditions. However, such sequences are derived from the nativenucleotide sequence on the basis that the native nucleotide sequence iscapable of being modified to exhibit a non-native sequence that stillencodes the same or substantially the same native amino acid sequence,or that the native amino acid sequence is capable of being used alongwith a codon table to back-translate, allowing the skilled artisan toarrive at a nucleotide sequence that encodes all or an insecticidalportion of a TIC900 or related protein. All of these sequences areintended to be within the scope of the present invention.

The B. thuringiensis strains containing a nucleotide sequence encoding aTIC900 or related protein and substantial equivalents thereof, can becultured using standard known media and fermentation techniques. Uponcompletion of the fermentation cycle, the bacteria expressing TIC900 ora homolog thereof can be harvested by first separating the B.thuringiensis spores and crystals from the spent fermentation broth bymeans well known in the art. The recovered B. thuringiensis spores andcrystals can be formulated into a wettable powder, a liquid concentrate,granules or other formulations by the addition of surfactants,dispersants, inert carriers and other components to facilitate handlingand application for particular target pests. The formulation andapplication procedures are all well known in the art. The proteins inthe spent fermentation broth including TIC900 or related proteins of thepresent invention can be concentrated and formulated into a wettablepowder, a liquid concentrate, granules or other formulations by theaddition of surfactants, dispersants, inert carriers and othercomponents to facilitate handling and application for particular targetpests.

Formulated bait granules containing an attractant and spores andcrystals of the B. thuringiensis isolates or concentrated spentfermentation media or insecticidal proteins purified from the spores orspent fermentation media, or recombinant microbes comprising thenucleotide sequences encoding TIC900 or related insecticidal proteinsobtainable from the B. thuringiensis isolates disclosed herein, can beapplied to the environment of the pest. The bait may be appliedliberally since the toxin does not affect animals or humans. Product mayalso be formulated as a spray or powder. Pests pick the product up ontheir feet or abdomen and carry it back to the nest where other pestswill be exposed to the toxin. The B. thuringiensis isolate orrecombinant host expressing a nucleotide sequence or gene encoding aTIC900 or related protein of the present invention may also beincorporated into a bait or food source for the pest.

As would be appreciated by a person skilled in the art, the pesticidalconcentration will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly. The pesticide will be present in at least 1% by weightand may be 100% by weight. The dry formulations will have from about1-95% by weight of the pesticide while the liquid formulations willgenerally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg or from about 5 to about 100 parts per million of the activecomponent insecticidal protein, i.e., the TIC900 protein, amino acidsequence variant thereof, insecticidal portion or fragment thereof, orhomolog thereof. These formulations will be administered at about 50 mg(liquid or dry) to 1 kg or more per hectare. The formulations can beapplied to the environment of the lepidopteran pests, e.g., plants,soil, or water by spraying, dusting, sprinkling, or the like, and canalso be applied to the surfaces of seeds as a seed treatment or seedcoating and can be permeated into the seed coat and/or cotyledon(s).

One skilled in the art would know that to achieve improved expression ofa Bt insecticidal protein in a plant, a nucleotide sequence encoding theBt protein, or an active variant or fragment of the protein, would firstneed to be prepared. Then the nucleotide sequence encoding the proteinor fragment thereof would be placed into an expression cassette thatfunctions in plants to cause the transcription of the coding sequenceinto a messenger RNA that is subsequently translated in the cells of theplant such that an insecticidally effective amount of the insecticidalprotein is produced within the plant tissues. One skilled in the artwould also know to transform a plant cell, preferably a corn, cotton,soybean, canola, rice, wheat, oat, grass, forage plant, cruciferousplant, fruit tree, ornamental flower, tomato, potato, carrot, kale, andtobacco plant cell and the like with the nucleotide sequence embeddedwithin the plant functional expression cassette, and to select for cellsthat contain the sequence and are expressing insecticidally effectiveamounts of the insecticidal protein, preferably a TIC900 or relatedprotein or insecticidal fragment thereof, and to produce plants fromsuch transformed cells. One skilled in the art would know to useelectroporation, infusion, ballistic methods, or Agrobacteriumtumefaciens mediated methods and the like for introducing the nucleotidesequences of the present invention or modifications thereof into a plantcell.

The term “variant or modified”, with reference to nucleotide sequences,is intended to refer to nucleotide sequences which encode the sametoxins or which encode equivalent toxins having similar insecticidalactivity, the term “equivalent toxin” referring to a toxin exhibitingthe same, essentially the same, or improved biological activity againstthe target pests as the claimed native or referent toxin. A variant ormodified nucleotide sequence intended for use in dicot plants wouldencode substantially the same amino acid sequence as the native codingsequence, i.e., the coding sequence found in nature, but would comprisea total combined GC composition from about 49 to about 58 percent, andwould utilize substantially the codon preference and codon usagefrequency determined by compiling such preference and usage frequenciesfrom a consortium of coding sequences derived from one or moreindividual dicot plant species intended to be transformed with thevariant or modified nucleotide sequence. A variant or modifiednucleotide sequence intended for use in a monocot plant would alsoencode substantially the same amino acid sequence as the native codingsequence, but would comprise a total combined GC composition from about52 to about 59 percent, and would also utilize substantially the codonpreference and codon usage frequency determined by compiling suchpreference and usage frequencies from a consortium of coding sequencesderived form one or more individual monocot plant species intended to betransformed with the variant or modified nucleotide sequence. Codonusage frequency is intended to refer to the number of times, on average,that a particular codon is used in a coding sequence. For a particularplant species, a codon that is intended to cause the incorporation of aparticular amino acid into a nascent amino acid sequence will beutilized on average with some relative fixed frequency. For amino acidsthat utilize only two codons, this frequency is generally aboutfifty-fifty, i.e., each codon being used about half the time, unless oneof the codons utilizes a substantially greater number of purines orpyrimidines that are not typically representative of the GC content ofthe particular plant species. For Bacillus species, for example, codingsequences generally are from about 60 to about 70 percent AT. Codonusage in Bacillus species is biased toward the use of codons that areenriched for the presence of A or T in a particular codon. Therefore,codons that primarily utilize G or C are used in a native and/ornaturally occurring Bacillus coding sequence with much less frequencythan codons that contain A's or T's. Therefore, when producing a variantor modified nucleotide sequence intended for use in a particular plant,monocot or dicot, it is important to ensure that appropriate attentionis given to the use of codons that are not particularly enriched withA's and T's where possible, and to avoid the incorporation of suspectedpolyadenylation sequences (see for example, U.S. Pat. No. 5,500,365).

As used herein, “synthetic coding sequences” or “non-naturally occurringcoding sequences” encoding the B. thuringiensis TIC900 proteins orhomologs or derivatives thereof as insecticidal toxins of the presentinvention are those prepared in a manner involving any sort of geneticisolation or manipulation. This includes isolation of the codingsequence from its naturally occurring state, manipulation of the codingsequence as by modification of the nucleotide coding sequence (asdescribed herein), chemical synthesis of all or part of a codingsequence using phosphoramidite chemistry and the like, or site-specificmutagenesis (as described herein), truncation of the coding sequence orany other manipulative or isolative method so that the amino acidsequence encoded by the non-naturally occurring coding sequence encodessubstantially the same insecticidal protein as the native codingsequence and furthermore exhibits substantially the same or an improvedlevel of insecticidal bioactivity as the native insecticidal toxinprotein.

As used herein, the phrase “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity. A sequence that is identical at every position in comparisonto a reference sequence is said to be identical to the referencesequence and vice-versa. A first nucleotide sequence when observed inthe 5′ to 3′ direction is said to be a “complement” of a second orreference nucleotide sequence observed in the 3′ to 5′ direction if thefirst nucleotide sequence exhibits complete complementarity with thesecond or reference sequence. As used herein, nucleic acid sequencemolecules are said to exhibit “complete complementarity” when everynucleotide of one of the sequences read 5′ to 3′ is complementary toevery nucleotide of the other sequence when read 3′ to 5′. A nucleotidesequence that is identical at every position when read 5′ to 3′ incomparison to a reference nucleotide sequence read 5′ to 3′ is said tobe identical to the reference sequence and vice-versa. A nucleotidesequence that is complementary to a reference nucleotide sequence willexhibit a sequence identical to the reverse complement sequence of thereference nucleotide sequence. These terms and descriptions are welldefined in the art and are easily understood by those of ordinary skillin the art.

As used herein, “substantial homology”, with reference to nucleic acidsequences, refers to nucleotide sequences that hybridize under stringentconditions to the TIC900 coding sequence as set forth in SEQ ID NO:3 orcomplements thereof. Sequences that hybridize under stringent conditionsto SEQ ID NO:3 or complements thereof, in particular from the nucleotidesequence from about nucleotide position 1 to about nucleotide position1806, and more particularly from about nucleotide position 121 to aboutnucleotide position 1806, contain one or more linear sequences that aresufficiently identical to one or more linear sequences of SEQ ID NO:3such that an alignment is able to take place and the two sequences arethen able, under stringent conditions, to form hydrogen bonds withcorresponding bases on the opposite strand to form a duplex moleculethat is sufficiently stable under the stringent conditions for a longenough period of time to be detectable using methods well known in theart. Such homologous sequences are from about 67% identical, to about70% identical, to about 80% identical, to about 85% identical, to about90% identical, to about 95% identical, to about 99% identical or greaterto the referent nucleotide sequence as set forth in SEQ ID NO:3 or thecomplement thereof. In addition, nucleotide sequences that encodeinsecticidal proteins isolatable from Bacillus thuringiensis strains andthe like, that hybridize under stringent conditions to SEQ ID NO:3 arealso envisioned to exhibit substantial homology with referent nucleotidesequences that hybridize under stringent conditions to the tic900 codingsequence as set forth in SEQ ID NO:3 or complements thereof. Suchnucleotide sequences are referred to herein as homologs of SEQ ID NO:3and the like and comprise SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:29, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQID NO:19, and related sequences and homologues thereof.

With reference to polypeptide sequences, the term “substantial homology”refers to polypeptides that are about 70% homologous to, about 80%homologous to, about 86% homologous to, about 90% homologous to, about95% homologous to, about 99% homologous to, a referent polypeptidesequence. More specifically, the inventors envision substantialhomologues to be about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, and 99 percent homologous to the referentpolypeptide sequence as set forth herein in SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:30, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, and SEQ ID NO:20.

With reference to the proteins of the instant application, the terms“variant amino acid sequence”, or “amino acid sequence variant”, or“modified amino acid sequence variant” are intended to refer to aminoacid sequences that are substantially equivalent to the amino acidsequences of the present invention. For example, a protein produced bythe introduction of a restriction site for convenience of molecularmanipulations into a coding sequence of the present invention thatresults in the addition or subtraction of one or more codons withoutotherwise (1) disrupting the native coding sequence, (2) disrupting thenative open reading frame, and (3) disrupting the insecticidalbiological activity of the protein, would constitute (a) a variant aminoacid sequence compared to the native insecticidal toxin, (b) an aminoacid sequence variant compared to the native insecticidal toxin, or (c)a modified amino acid sequence variant compared to the nativeinsecticidal toxin. One skilled in the art would recognize that thereare other types of modifications that can be made to the amino acidsequence of the present invention without disrupting the biologicalactivity of the protein. Insertions, deletions, and substitutions arewithin the scope of the present disclosure to the extent that theresulting amino acid sequence variant exhibits insecticidal activity noless than that of the native insecticidal protein. Chimeras of theproteins disclosed herein, fusions of the proteins or parts of theproteins disclosed herein, and permuteins of the proteins disclosedherein are specifically contemplated.

The inventors contemplate that the protein compositions disclosed hereinwill find particular utility as insecticides for topical and/or systemicapplication to field crops, grasses, fruits and vegetables, andornamental plants. In a preferred embodiment, the bioinsecticidecomposition comprises an oil flowable suspension of bacterial cells thatexpresses a novel insecticidal protein disclosed herein. Preferably thecells are B. thuringiensis EG5438 or SIC9002 cells, however, any suchbacterial host cell expressing the novel nucleic acid segments disclosedherein and producing a crystal protein is contemplated to be useful,such as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.

In another embodiment, the bioinsecticide composition comprises a waterdispersible granule. This granule comprises bacterial cells that expressa novel insecticidal protein disclosed herein. Preferred bacterial cellsare B. thuringiensis EG5438 or SIC9002 cells, however, bacteria such asB. megaterium, B. subtilis, E. coli, or Pseudomonas spp. cellstransformed with a DNA segment disclosed herein and expressing theinsecticidal protein are also contemplated to be useful.

In a third embodiment, the bioinsecticide composition comprises awettable powder, dust, pellet, or collodial concentrate. This powdercomprises bacterial cells that express a novel insecticidal proteindisclosed herein. Preferred bacterial cells are B. thuringiensis EG5438or SIC9002 cells, however, bacteria such as B. megaterium, B. subtilis,E. coli, or Pseudomonas spp. cells transformed with a DNA segmentdisclosed herein and expressing the insecticidal protein are alsocontemplated to be useful. Such dry forms of the insecticidalcompositions may be formulated to dissolve immediately upon wetting, oralternatively, dissolve in a controlled-release, sustained-release, orother time-dependent manner.

In a fourth embodiment, the bio-insecticide composition comprises anaqueous suspension of bacterial cells such as those described above thatexpress the insecticidal protein. Such aqueous suspensions may beprovided as a concentrated stock solution which is diluted prior toapplication, or alternatively, as a diluted solution ready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the insecticidal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise intact B. thuringiensiscells expressing the protein of interest, such bacteria may beformulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (phyllosilicates, carbonates, sulfates,phosphates, and the like) or botanical materials (powdered corncobs,rice hulls, walnut shells, and the like). The formulations may includespreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel TIC900 or TIC900-derived or related protein orhomolog thereof may be prepared by native or recombinant bacterialexpression systems in vitro and isolated for subsequent fieldapplication. Such protein may be either in crude cell lysates,suspensions, colloids, etc., or alternatively may be purified, refined,buffered, and/or further processed, before formulating in an activebiocidal formulation. Likewise, under certain circumstances, it may bedesirable to isolate the protein in some crystalline form and/or asspores from bacterial cultures expressing the insecticidal protein andapply solutions, suspensions, or collodial preparations of such crystalsand/or spores as the active bioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) are applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificlepidopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating thebacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target lepidopteran insect, typically onto thefoliage of the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well known to those ofskill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition that is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bio-insecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 1% to about 99% or more by weight ofthe composition, while liquid formulations may generally comprise fromabout 1% to about 99% or more of the active ingredient by weight.Formulations that comprise intact bacterial cells will generally containfrom about 10⁴ to about 10¹² cells/mg.

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 50g to about 500 g of active ingredient, or of from about 500 g to about1000 g, or of from about 1000 g to about 5000 g or more of activeingredient.

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. In particular embodiments of the invention,amino acid sequence variants of the proteins of the present inventionare contemplated to be useful for increasing the insecticidal activityof the protein, and consequently increasing the insecticidal activityand/or expression of the recombinant transgene in a plant cell. Theamino acid changes may be achieved by changing the codons of the DNAsequence.

Proteins that are substantially equivalent to the proteins of theinstant application are intended to be biologically functionallyequivalent. As used herein, the phrase “biological functionalequivalents”, with respect to the insecticidal proteins of the presentinvention, are peptides, polypeptides and proteins that contain asequence or moiety exhibiting sequence similarity to the novel peptidesof the present invention, such as a TIC900 or related protein orinsecticidal fragment thereof, and that exhibit the same or similarfunctional properties as that of the polypeptides disclosed herein,including insecticidal activity. Biological equivalents also includepeptides, polypeptides and proteins that react with, i.e., specificallybind to antibodies raised against epitopes present on or within TIC900and related proteins and that exhibit the same or similar binding orreactive activity, including both monoclonal and polyclonal antibodies.

It is also contemplated that the proteins of the present invention couldbe useful for protecting dicot plants from insect infestation. Suchinfestations could be the result of lepidopteran, coleopteran, dipteran,or even infestation by mites, mealworms, grubs, or a wide variety ofinsects that injure the plant by piercing the plant tissues andextracting the nutrients intended for plant growth and development.Modifications to the primary amino acid sequence of the proteins of thepresent invention could result in a protein that exhibits a host rangedifferent from that of the native protein.

The proteins of the present invention, because of their localizationinto the extracellular space when expressed by Bacillus strains, may beuseful for targeting other proteins for localization into theextracellular space. For example, the skilled artisan would know to linka first protein that is not normally secreted into the extracellularspace to a second protein that is normally secreted into theextracellular space in order to achieve the localization of the firstprotein into the extracellular space. The proteins of the presentinvention could be fused by any number of means well known in the art toone or more insecticidal toxins such as crystalline delta-endotoxins toform a chimeric protein that is targeted for secretion into theextracellular space surrounding a particular host cell. It is evenenvisioned that the secretion event itself could lead to the separationof the two protein parts such that two separate and distinctinsecticidal proteins are released into the extracellular spacesurrounding a particular host cell. The two proteins could either (1)both be toxic to the same insect species but effectuate theirinsecticidal activity using different modes of action, or (2) each betoxic to different insect species. It is conceivable that any number ofinsecticidal proteins could be linked end to end to the proteins of thepresent invention to form multimeric chimeras that are targeted to theextracellular space surrounding a particular host cell. It ispreferable, in situations in which it is contemplated that other Btinsecticidal proteins are used, that the insecticidal proteins fused tothe proteins of the present invention be less than full length Cry1proteins, more preferably merely core insecticidal toxin fragments ofCry1 proteins, Cry2A proteins, Cry3 proteins, Cry9 proteins, etc. Such“other” proteins conceivably could be green fluorescent and relatedproteins and variants, kinases and phosphatases for modulating cellsignaling processes, nucleases, lipases, herbicide tolerance proteinsexpressed from genes such as gox, various epsps homologues, bar andhomologues and the like, PhnO, NptII, Aad, and the like. All of theseproteins could be used as selectable markers as well, particularly whenlinked to a gene encoding one or more of the proteins of the presentinvention, to track the presence of the genes encoding one or more ofthe proteins of the present invention in a plant or other host cell.

The proteins of the present invention could be targeted for import intoa subcellular organelle. For example, a first nucleotide sequenceencoding a chloroplast or plastid targeting sequence could be operablylinked or fused to a second nucleotide sequence encoding an insecticidalprotein of the present invention to produce a chimeric precursor proteinthat is targeted for insertion into the chloroplast or plastid within aplant cell. Expression of such chimeric proteins would result in theimport of the proteins of the present invention into the plantchloroplast or plastid, resulting in the localization of theinsecticidal toxin or insecticidal fragment thereof into the chloroplastor plastid. Additionally, a nucleotide sequence encoding one or moreproteins of the present invention could be localized to the chloroplastor plastid for expression. The localization of the nucleotide sequencesto the plastid or chloroplast could result in the incorporation of thenucleotide sequences into the chloroplast or plastid genome, or couldresult in the presence of an autonomously replicating nucleic acidsequence encoding the protein of the present invention. In either sense,the proteins of the present invention would be localized to thechloroplast or plastid. As used herein therefore, the phrase“chloroplast or plastid localized” refers to a biological molecule,either polynucleotide or polypeptide, which is positioned within thechloroplast or plastid such that the molecule is isolated from thecellular cytoplasmic milieu, and functions within the chloroplast orplastid cytoplasm to provide the beneficial insecticidal effects claimedin the instant invention. Localization of a biological molecule to thechloroplast or plastid can occur, with reference to polynucleotides, byartificial mechanical means such as electroporation, mechanicalmicroinjection, or by polynucleotide coated microprojectile bombardment,or with reference to polypeptides, by secretory or import means whereina natural, synthetic, or heterologous plastid or chloroplast targetingpeptide sequence is used which functions to target, insert, assist, orlocalize a linked polypeptide into a chloroplast or plastid. In anyevent, localization of one or more insecticidal proteins to thechloroplast or plastid necessarily implies that the resulting plantcontaining cells which contain plastids that contain such insecticidalprotein or proteins localized within must also exhibit normalmorphological characteristics. It is not known which, if any,insecticidal protein when localized to the chloroplast or plastid, willresult in the achievement of a recombinant plant exhibiting normalmorphological characteristics exemplified without limitation by anabsence of chlorosis, an absence of stunted or stunting of the plantphysiology including but not limited to thicker than average stalks,shortened stalks or internodes, inappropriate flowering, infertility,decreased yield, etc.

As used herein, the phrase “operatively linked” or “operably linked”refers to nucleic acid coding segments connected in frame so that theproperties of one influence the expression of the other. These phrasesand groups of words can also be used to refer to amino acid sequenceswhich exhibit some function when linked to another amino acid sequence,for example, a signal peptide when linked to a protein of interest isreferred to as being operably linked to the protein of interest for thepurpose of targeting the protein of interest to the secretory apparatusof the host cell in which the protein is produced.

For the purposes of the present invention, the word “gene” refers to anucleotide sequence that contains an open reading frame encoding aTIC900 protein, or an insecticidal fragment thereof, or an amino acidsequence variant thereof, or a related protein homolog or insecticidalfragment thereof or amino acid sequence variant thereof that is at leastoperably linked to a promoter sequence and a transcription terminationsequence, wherein the promoter and transcription termination sequencesare functional in the host cell in which the protein is produced. Asused herein, “structural gene” refers to a gene that is expressed toproduce a polypeptide. A structural gene of the present invention cancontain, in addition to promoter and transcription terminationsequences, five prime non-translated sequences, intronic sequences, andenhancer elements that function in plants in particular, and preferablythose that are derived from monocotyledonous plants such as maize plantsor from dicotyledonous plants such tobacco plants or cruciferousvegetable plants that, when linked together in proper sequence with oneor more coding sequences of the present invention result in improvedlevels of expression in particular plant tissues, and preferably resultin enhanced expression in leaves and stem tissues of those plants.

Nucleotide sequence information provided by the present invention allowsfor the preparation of relatively short DNA sequences, referred toherein as probes or primers, having the ability to specificallyhybridize to sequences of the selected polynucleotides disclosed herein.Such nucleic acid probes of an appropriate length are prepared based ona consideration of selected polypeptide sequences encoding theinsecticidal polypeptides of the present invention, e.g., a sequencesuch as that shown in all or a probe specific part of SEQ ID NO:3, allor a probe specific part of SEQ ID NO:5, all or a probe specific part ofSEQ ID NO:7, all or a probe specific part of SEQ ID NO:9, all or a probespecific part of SEQ ID NO:29, all or a probe specific part of SEQ IDNO:11, all or a probe specific part of SEQ ID NO:13, all or a probespecific part of SEQ ID NO:15, all or a probe specific part of SEQ IDNO:17, all or a probe specific part of SEQ ID NO:19, and the like.Reference to the phrase “all or a probe specific part of” is intended torefer to a nucleotide sequence probe comprising at least from about 15to about 50, more or less, contiguous nucleotides selected from thegroup of nucleotides set forth in a particular referent sequence such asSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19. Theability of such nucleic acid probes to specifically hybridize to anucleotide sequence encoding an insecticidal polypeptide sequence lendsto them particular utility in a variety of embodiments. Mostimportantly, the probes may be used in a variety of assays for detectingthe presence of complementary sequences in a given biological sample. Byreference to the term “biological sample”, it is intended that anysample that contains a referent nucleotide sequence that can be detectedby a probe sequence as set forth herein is a sample that contains abiological molecule selected from the group consisting of contiguousnucleotide sequences set forth herein, and therefore the sample is thusreferred to as a “biological sample”.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or modifying adefined segment of an insecticidal protein coding sequence from B.thuringiensis or from Bacillus sphaericus and the like using thermalamplification technology. Segments of nucleotide sequences related tothe polynucleotides encoding the insecticidal polypeptides of thepresent invention may also be isolated and characterized using thermalamplification technology and such primers.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays or as a primer includes sequences that arecomplementary to at least a 14 to 30 or more contiguous stretch ofnucleotides of a polynucleotide sequence encoding all or a part of aninsecticidal protein of the present invention, such as that shown in SEQID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:29, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, and SEQ ID NO:22.

A primer or probe size of at least 14 nucleotides in length helps toensure that the fragment will be of sufficient length to form a duplexmolecule that is both stable and selective. Molecules havingcomplementary sequences over segments greater than 14 bases in lengthare generally preferred. In order to increase stability and selectivityof the hybrid, and thereby improve the quality and degree of specifichybrid molecules obtained, one will generally prefer to design nucleicacid molecules having tic900-complementary sequences and the like of 14to 20 nucleotides, or even longer where desired. Such fragments may bereadily prepared by, for example, directly synthesizing the fragment bychemical means, by application of nucleic acid reproduction technology,or by excising selected DNA fragments from recombinant sequenceslocalized in plasmids or other vectors containing appropriate insertsand suitable restriction sites.

The present invention also contemplates an expression vector comprisinga polynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to a coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region. The coding region mayinclude a segment encoding a B. thuringiensis insecticidal toxin of thepresent invention and a segment encoding a chloroplast or plastidtargeting peptide. The DNA molecule comprising the expression vector mayalso contain a functional intron sequence positioned either upstream ofthe coding sequence or even within the coding sequence, and may alsocontain a five prime (5′) non-translated leader sequence (i.e., a UTR or5′-UTR) positioned between the promoter and the point of translationalinitiation.

As used herein and with reference to promoter elements, the terms“operatively linked” or “operably linked” are intended to indicate thata nucleotide sequence that contains a promoter, i.e. a genetic elementthat functions in a particular host cell to drive the initiation oftranscription, is connected to a coding region in such a way that thetranscription of that coding region is controlled and substantiallyregulated by that promoter. Means for operatively linking a promoter toa coding region are well known in the art. Promoters that function inbacteria are well known in the art. Exemplary and preferred promotersfor the B. thuringiensis crystal proteins include the sigA, sigE, andsigK gene promoters. Alternatively, native, modified, heterologous, orrecombinant promoters derived from Bacillus thuringiensis or otherBacillus species can be used for achieving expression of the proteins ofthe present invention in a Bacillus species strain.

Where a nucleotide sequence encoding all or an insecticidal part of aprotein of the present invention is to be used to transform a plant, apromoter is selected that has the ability to drive expression of thecoding sequence in that particular species of a plant. Promoters thatfunction in different plant species are also well known in the art.Promoters useful for expression of polypeptides in plants are those thatare inducible, viral, synthetic, or constitutive as described in Odellet al. (Nature 313:810-812, 1985), and/or promoters that are temporallyregulated, spatially regulated, and spatio-temporally regulated.Preferred promoters include the enhanced CaMV35S promoters, the GBOX10promoter, the FMV35S promoter, the rice Actin promoter, and variants andchimeras thereof. For optimum control of ECB species by expression ofthe proteins of the present invention in plants, for example, it ispreferable to achieve the highest levels of expression of these proteinswithin the leaves and stems of maize plants. Substantial temporal orspatial regulation refers to the expression of a gene within a plant orplant tissue from a plant operable promoter. With reference to temporalregulation, a promoter may be regulated for expression only duringspecific times during plant cell or tissue or even whole plant growthand development. A promoter that is actively expressing one or moregenes only during seed germination would be one example of temporalregulation. Other examples could include promoters that are activelyexpressing one or more genes only during times when the plant, plantcell or plant tissue is exposed to certain light intensities or duringtotal darkness. Substantial temporal regulation refers to a promoterwhich is actively expressed at a certain time but which may or may notbe completely suppressed at other times, such that expression may stillbe detected by monitoring for the presence of some indicator such as anenzyme produced from a coding sequence linked to such a promoter, or asmeasured by the increase or decrease in some gene products such as anmRNA produced at various times throughout plant growth, differentiation,and development and/or in response to various environmental stimuli.Substantial spatial regulation refers to the expression of a gene linkedto a promoter from which expression proceeds only during growth anddevelopment of certain cells or tissues within a plant. For example, atapetal promoter is one that is substantially spatially expressed duringflower growth and development. Similarly, a leaf specific or leafenhanced promoter would only be expected to be substantially spatiallyexpressed from within leaf cells or leaf tissues. Substantiallyspatially regulated also refers to the level of expression from aparticular tissue specific promoter in that particular tissue and asrelated to levels of expression from that or a similar promoter in othertissues, wherein expression may also be detected in tissues other thanthe particular tissue in which the promoter expression is preferred, butat significantly lower expression levels as measured by the productionof an enzyme produced from a coding sequence linked to the promoter orby the appearance of some detectable gene product. Promoters can also beboth substantially temporally and substantially spatially regulatedtogether and simultaneously in a coordinately regulated manner. Otherpromoters specifically intended to be within the scope of the presentinvention include but are not limited to the ubiquitin promoter, thesugarcane bacilliform DNA virus promoter, the ribulose bis-phosphatecarboxylase large subunit promoter, among others.

Preferred intron sequences for achieving optimum expression ofnon-naturally occurring nucleotide sequences in monocotyledonous plantsmay also be included in the DNA expression construct. Such an intron istypically placed near the 5′ of the mRNA within or immediatelydownstream of an untranslated sequence. The intron could be obtainedfrom, but not limited to, a set of introns consisting of the maize HeatShock Protein (HSP) 70 intron (U.S. Pat. No. 5,424,412; 1995), the riceAct1 intron (McElroy et al., Plant Cell 2:163-171, 1990), the Adh intron1 (Callis et al., Genes & Develop. 1:1183-1200, 1987), or the sucrosesynthase intron (Vasil et al., Plant Phys. 91:1575-1579, 1989).

Another element that functions to regulate or to modulate geneexpression is the DNA sequence between the transcription initiation siteand the start of the coding sequence, termed the untranslated leadersequence (UTL). Compilations of leader sequences have been made topredict optimum or sub-optimum sequences and generate “consensus” andpreferred leader sequences (Joshi, Nucl. Acids Res. 15:9627-9640, 1987).Preferred leader sequences are contemplated to include those thatcomprise sequences predicted to direct optimum expression of the linkedstructural gene, i.e. to include a preferred consensus leader sequencethat increases or maintains mRNA stability and prevents inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat from genes that are highly expressed in plants, and in particularin maize will be most preferred. One particularly useful leader is thepetunia HSP70 leader.

Transcription enhancers or duplications of enhancers could be used toincrease expression. These enhancers often are found 5′ to the start oftranscription in a promoter that functions in eukaryotic cells, but canoften be inserted in the forward or reverse orientation 5′ or 3′ to thecoding sequence. Examples of enhancers include elements from the CaMV35S promoter, octopine synthase genes (Ellis et al., EMBO Journal6:11-16, 1987), the rice actin gene, and promoter from non-planteukaryotes (e.g., yeast; Ma et al., Nature 334:631-633, 1988).

RNA polymerase transcribes a nuclear genome DNA coding sequence througha site where polyadenylation occurs. Typically, DNA sequences located afew hundred base pairs downstream of the polyadenylation site serve toterminate transcription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of nuclear transcribed messenger RNA (mRNA).For coding sequences introduced into a chloroplast or plastid, or into achloroplast or plastid genome, mRNA transcription termination is similarto methods well known in the bacterial gene expression art. For example,either in a polycistronic or a monocistronic sequence, transcription canbe terminated by stem and loop structures or structures similar tobacterial rho dependent sequences.

Expression constructs will typically include a coding sequenceexemplified in the present invention or a derivative thereof along witha 3′ end DNA sequence that functions as a signal to terminatetranscription and, in constructs intended for expression from the plantnuclear genome, allow for the 3′ end polyadenylation of the resultantRNA transcript. The most preferred 3′ elements are contemplated to bethose from the nopaline synthase gene of A. tumefaciens (nos 3′ end),the terminator for the T7 transcript from the octopine synthase gene ofA. tumefaciens, and the pea RUBISCO synthase E9 gene (E9 3′) 3′non-translated transcription termination and polyadenylation sequence.These and other 3′ end regulatory sequences are well known in the art.

Preferred plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g.,by Herrera-Estrella (Nature 303:209-213, 1983), Bevan (Nature304:184-187, 1983), Klee (Bio/Technol. 3:637-642, 1985).

The present invention discloses isolated and purified nucleotidesequences encoding insecticidal proteins derived from Bacillus species,and particularly from Bacillus thuringiensis species. In particular, theB. thuringiensis strains EG5438, EG3879, EG4332, EG4971, EG4090, EG4293,EG4611, EG5526, EG5023 and EG4092 are each shown herein to produce oneor more soluble insecticidal proteins that are localized to culturesupernatants (see Table 1).

TABLE 1 TIC900 Related Proteins and Source B. thuringiensis StrainsSource Bt Strain TIC900 Related Protein EG3879, EG5526 TIC402, (TIC964)*EG4332 TIC403 EG4971 TIC404 EG4611 TIC434 EG4090 TIC961 EG4293 TIC962EG4611 TIC963 EG5438^(#) TIC900 EG5023 TIC965 EG4092 TIC966 *the aminoacid sequence of TIC964, obtained from strain EG5526, was deduced afternucleotide sequence analysis of a gene exhibiting homology to tic900,and was determined to be identical to tic402 obtained from strainEG3879. ^(#)signifies that this strain has been deposited underconditions that assure access to the culture to authorized partiesduring the pendency of this patent application or patents issuedtherefrom.

The B. thuringiensis strains and other bacterial strains describedherein may be cultured using conventional growth media and standardfermentation techniques. The B. thuringiensis strains harboring one ormore tic900 or related genes may be fermented as described herein untilthe cultured B. thuringiensis cells reach the stage of their growthcycle when the TIC900 and/or related proteins are produced.

Subject cultures have been deposited under conditions that assure thataccess to the culture will be available to authorized parties during thependency of this patent application or patents issued. However, itshould be understood that the availability of a deposit does notconstitute a license to practice the subject invention in derogation ofpatent rights granted by governmental action.

TIC900 and related proteins of the present invention are produced asshown herein and secreted into the growth media during the vegetativephase of growth. Fermentations using the strains of the presentinvention may be continued through the sporulation stage when crystalproteins, if any, are formed along with the spores. The spores and celldebris can be separated from the supernatant by centrifugation, and thespent culture medium can be used to isolate the insecticidal proteins ofthe present invention. The inventors herein illustrate the method ofammonium sulfate precipitation as one means for concentrating andcollecting all or most of the proteins present in the spent andclarified culture medium. However, one skilled in the art will recognizethat there are a number of other means available for purifying andisolating the proteins of the present invention. Gel filtration and sizeexclusion chromatography are two readily available means for extractingproteins directly from the spent media. Spent media can also be desaltedand the filtrate used to extract protein using ion exchange columns.Also, affinity columns, containing antibodies that bind specifically toTIC900 or related proteins can be used to purify the proteins of thepresent invention directly from the media.

The amino acid sequences of the present invention have been compared tothe amino acid sequences present in commercially available proteinsequence databases, and no significant homologies or similarities havebeen identified. Based on this analysis, the TIC900 protein and relatedsequences appear to be unique and form the basis for the establishmentof a new and separate class of Bacillus insecticidal proteins becausethe proteins of the present invention do not exhibit any relationship toother known insecticidal proteins.

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments that encode them and still obtaina functional molecule that encodes a protein or peptide with desirablecharacteristics. The biologically functional equivalent peptides,polypeptides, and proteins contemplated herein should possess from about70% or greater sequence similarity, or from about 80% or greatersequence similarity, or from about 90% or greater sequence similarity,to the sequence of, or corresponding moiety within, the fundamentalTIC900 amino acid sequence as set forth in SEQ ID NO:4, or thecorresponding moiety within the amino acid sequences as set forth in SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:30, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20 and relatedsequences.

According to the present invention reference to the tic900 gene andencoded protein toxin, includes not only the full length sequencesdisclosed herein but also fragments of these sequences, naturalvariants, mutants, and recombinant or genetically engineered derivativesof the tic900 gene comprising SEQ ID NO:3. Such encoded proteins shouldretain essentially the same as or greater characteristic insecticidalproperties than those of the TIC900 protein comprising SEQ ID NO:4. Theproteins useful in the present invention may also include fusionproteins that retain the characteristic insecticidal propertiesessentially the same as or greater than those of the TIC900 protein. Insome instances, the fusion protein may contain, in addition to thecharacteristic insecticidal properties of the proteins specificallyexemplified herein, another insecticidal activity contributed by theamino acid sequence of the fusion partner. Alternatively,crystallographic analysis of the TIC900 protein or insecticidal variantsthereof may provide a means for determining whether the protein would bea candidate for the construction of a permutein that exhibits the sameor preferably greater insecticidal activity than the native TIC900 orrelated protein, and which preferably exhibits improved characteristicsrelated to expression in a preferred host cell such as a plant cell.

It should be apparent to a person skilled in the art that nucleotidesequences encoding lepidopteran inhibitory toxins can be identified andobtained through several means. The specific sequences exemplifiedherein may be obtained from the isolates deposited at a culturedepository as described above. These sequences, or portions or variantsthereof, may also be constructed synthetically, for example, by use of anucleotide sequence synthesizer. Variations of coding sequences may bereadily constructed using standard techniques for making pointmutations. Also, fragments of these sequences can be made usingcommercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as Bal31 or site-directedmutagenesis may be used to systematically excise nucleotides from theends of such sequences as exemplified herein or from within the proteincoding sequence. Also, nucleotide sequences that encode insecticidallyactive protein fragments may be obtained using a variety of restrictionenzymes, endonucleases, thermal amplification methods, and the like.Proteases such as proteinase K, trypsin, chymotrypsin, pepsin, and thelike may be used to directly obtain active fragments of these toxins.

Other toxins and nucleotide sequences encoding such toxins related tothe toxins and coding sequences of the present invention can be derivedfrom DNA obtained from B. thuringiensis, B. laterosporous, B.sphaericus, and related Bacillus species isolates using the teachingsprovided in the art in combination with the nucleotide sequencesdisclosed herein. Such toxins and nucleotides sequences that are relatedto the toxins and coding sequences of the present invention are deemedherein to be equivalent to the toxins and nucleotide sequences of thepresent invention. By “equivalent” it is meant that a protein exhibitsthe characteristics of the TIC900 protein, including but not limited tosimilar insecticidal inhibitory bioactivity, host range of insecticidalbioactivity, exhibits similar antigenic epitopes that cross react withantibodies raised against TIC900 and related proteins, exhibit a similarsize relative to TIC900 and related proteins, exhibit similar expressionprofiles and characteristics, exhibit a propensity for seclusion to theextracellular environment when expressed in Bacillus thuringiensis orrelated bacterial species, and the like. The phrase “exhibit apropensity for seclusion to the extracellular environment” is intendedto include TIC900 and related proteins including but not limited toTIC402, TIC403, TIC404, TIC434, TIC961, TIC962, TIC963, TIC965 andTIC966 that are produced by the bacterium or host cell as a precursorprotein that contains an amino acid sequence linked to the insecticidalprotein that functions to target the insecticidal protein to a bacterialor host cell secretory apparatus and which, upon contact with thesecretory apparatus, is proteolytically cleaved by a signal peptidase,releasing the mature or insecticidal protein into the extracellularenvironment in the case of a gram positive microbe, at least into theperiplasm in the case of a gram negative microbe, and into theendoplasmic reticulum or secretory vesicle or into a subcellularorganelle such as a mitochondria or chloroplast or plastic in the caseof a fungal or plant or other eukaryotic host cell.

There are a number of methods for identifying the presence of andobtaining equivalent insecticidal toxins related to the peptidesdisclosed herein. For example, antibodies to the insecticidal toxinsdisclosed and claimed herein can be used to identify and isolate othertoxins from a mixture of proteins. Specifically, antibodies may beraised to the portions of the toxins that are most constant within thenew class of proteins and most distinct from other B. thuringiensistoxins. These antibodies can then be used to specifically identifyequivalent toxins with the characteristic activity byimmuno-precipitation, enzyme linked immuno-sorbent assay (ELISA), orWestern blotting. Antibodies to the toxins disclosed herein, or toequivalent toxins, or fragments of these toxins, can readily be preparedusing standard procedures in the art. The nucleotide sequences thatencode these toxins can then be obtained from the microorganism or othervarious sources.

Fragments and equivalents that retain the insecticidal activity of theexemplified toxins would be within the scope of the present invention.Also, because of the redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate these alternative DNA sequences encoding the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe present invention.

It is well known in the art that certain amino acids may be substitutedfor other amino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated by theinventors that various changes may be made in the peptide sequences ofthe compositions disclosed herein, or corresponding DNA sequences whichencode said peptides without appreciable loss of their biologicalutility or activity. Such substitutions are also known in the art asconservative substitutions.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein (U.S. Pat. No. 4,554,101).

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take the variousforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Peptides, polypeptides, and proteins biologically functionallyequivalent to TIC900, TIC402, TIC403, TIC404, TIC434, TIC961, TIC962,TIC963, TIC965 and TIC966 include amino acid sequences containingconservative amino acid changes in the fundamental sequence shown in SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:30, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:20. Insuch amino acid sequences, one or more amino acids in the fundamentalsequence is (are) substituted with another amino acid(s), the charge andpolarity of which is similar to that of the native amino acid, i.e. aconservative amino acid substitution, resulting in a silent change.

Substitutes for an amino acid within the fundamental polypeptidesequence can be selected from other members of the class to which thenaturally occurring amino acid belongs Amino acids can be divided intothe following four groups: (1) acidic amino acids; (2) basic aminoacids; (3) neutral polar amino acids; and (4) neutral non-polar aminoacids. Representative amino acids within these various groups include,but are not limited to: (1) acidic (negatively charged) amino acids suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cyteine, cystine, tyrosine,asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

Conservative amino acid changes within the fundamental polypeptidesequences of the present invention can be made by substituting one aminoacid within one of these groups with another amino acid within the samegroup. Biologically functional equivalents of TIC900 and relatedsequences can have 10 or fewer conservative amino acid changes, morepreferably seven or fewer conservative amino acid changes, and mostpreferably five or fewer conservative amino acid changes. The encodingnucleotide sequence (gene, plasmid DNA, cDNA, or synthetic DNA) willthus have corresponding base substitutions, permitting it to encodebiologically functional equivalent forms of TIC900.

Amino acid sequence variants of TIC900 and related sequences can be madeby procedures well known in the art.

A further method for identifying the toxins and genes of the presentinvention is through the use of oligonucleotide probes. These probes areessentially nucleotide sequences that hybridize under stringenthybridization conditions to the TIC900 coding sequence or a sequencerelated to a TIC900 coding sequence. As is well known in the art, if aprobe molecule and nucleic acid sequence molecule in a sample hybridizeby forming a strong enough bond between the two molecules, it can bereasonably assumed that the two molecules exhibit substantial homology.Probe binding is detected using any number of means known in the artincluding but not limited to fluorescence, luminescence, isotopic,immunological, surface plasmon resonance spectroscopy, and the like.Such probe analysis provides a rapid method for identifyingtoxin-encoding genes of the present invention. The nucleotide segmentsthat are used as probes according to the invention can be synthesized byuse of DNA synthesizers using standard procedures or by other meansknown in the art. These nucleotide sequences can also be used as PCRprimers to amplify nucleotide sequences of the present invention orportions thereof.

The tic900 and related nucleotide coding sequences as set forth hereinin SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19 maybe used as hybridization probes to identify and isolate natural variantsof the tic900 and related nucleotide coding sequences from other strainsof B. thuringiensis or from other microorganisms. The present inventionencompasses nucleotide sequences from microorganisms, where thenucleotide sequences are isolatable by hybridization with all, or part,of the Bacillus nucleotide sequence of the invention. Proteins encodedby such nucleotide sequences can be tested for insecticidal activity.The invention also encompasses the proteins encoded by the nucleotidesequences.

Antibodies to TIC900 or related proteins of the present invention may beproduced using standard immunological techniques for production ofpolyclonal antisera and, if desired, immortalizing theantibody-producing cells of the immunized host for sources of monoclonalantibody production. Techniques for producing antibodies to anysubstance of interest are well known, e.g., as in Harlow and Lane (1988)and as in Goding (1986). The anti-TIC900 antibodies may be used asprobes to identify B. thuringiensis strains or other microorganisms thatproduce variants of TIC900 or related proteins that are encoded byvariations of a tic900 or related gene. The present inventionencompasses proteins obtained from organisms wherein the proteinsobtained cross-react with antibodies raised against one or more of theproteins of the present invention.

The antibodies produced in the present invention are also useful inimmunoassays for determining the amount or presence of a TIC900 orrelated protein. Such assays are also useful in quality-controlledproduction of compositions containing TIC900 or related proteins of thepresent invention. In addition, the antibodies can be used to assess theefficacy of recombinant production of a TIC900 or related protein, aswell as for screening expression libraries for the presence of TIC900 orrelated protein coding sequences. Antibodies are useful also as affinityligands for purifying and/or isolating TIC900 and related proteins.TIC900 and related antigenic epitopes may be obtained by over expressingfull or partial lengths of a sequence encoding all or part of a TIC900or related protein in a preferred host cell.

The peptides of the present invention are primarily, though notexclusively, intended for use in plants, and in certain preferredembodiments, nucleotide sequences modified for encoding the proteins ofthe present invention in plants are contained within one or more plasmidvectors. Such vectors may contain a variety of regulatory and otherelements intended to allow for optimal expression of the proteins of thepresent invention in plant cells. These additional elements may includepromoters, terminators, and introns as outlined above. Any vectorcontaining the DNA construct and any regulatory or other elements may beselected from the group consisting of a yeast artificial chromosome,bacterial artificial chromosome, a plasmid, or a cosmid, and the like.Further, the expression vectors themselves may be of a variety of forms.These forms may differ for various reasons, and will likely be comprisedof varying components depending upon whether they are intended totransform a monocotyledonous plant or a dicotyledonous plant.

Vectors further envisioned to be within the scope of the presentinvention include those vectors capable of containing a tic900 orrelated nucleic acid compositions disclosed above, as well as any otherDNA constructs which further comprise plant-expressible coding regionsfor other insecticidal proteins derived from Bacillus species.

The nucleotide sequence encoding the TIC900 insecticidal protein (SEQ IDNO:4) or encoding a related polypeptide sequence such as TIC402 (SEQ IDNO:6), TIC403 (SEQ ID NO:8), TIC404 (SEQ ID NO:10), TIC434 (SEQ IDNO:30), TIC961 (SEQ ID NO:12), TIC962 (SEQ ID NO:14), TIC963 (SEQ IDNO:16), TIC965 (SEQ ID NO:18) and TIC966 (SEQ ID NO:20) may beintroduced into a variety of microorganism hosts without undueexperimentation, using procedures well known to those skilled in the artof transforming suitable hosts under conditions which allow for stablemaintenance and expression of the cloned genes (Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual 2^(nd) Ed., Cold Spring HarborPress, New York). Suitable hosts that allow for expression of the TIC900protein (SEQ ID NO:4) and related sequences include B. thuringiensis andother Bacillus species such as Bacillus subtilis or Bacillus megaterium.Genetically altered or engineered microorganisms containing the tic900gene (SEQ ID NO:3) can also contain nucleotide sequences encoding othertoxin proteins present in the same microorganism; these coding sequencescould concurrently produce insecticidal proteins different from theTIC900 or related proteins. In particular, it would be preferable toproduce two or more different insecticidal proteins in a host cell,wherein each protein is toxic to the same insect species and eachprotein exhibits a mode of action different from the other(s).

Plant-colonizing or stem-colonizing microorganisms may also be employedas host cells for the production of a TIC900 or related protein.Exemplary microorganism hosts for B. thuringiensis toxin genes includethe plant-colonizing microbe Clavibacter xyli as described by Turner etal. (1993; Endophytes: an alternative genome for crop improvement;International crop science I. International Crop Science Congress, Ames,Iowa, USA, 14-22 Jul. 1992, pp. 555-560).

The toxin-encoding nucleotide sequences obtainable from the isolates ofthe present invention can be introduced into a wide variety of microbialor plant hosts. Expression of the toxin gene results, directly orindirectly, in the intracellular production and maintenance of thepesticide. With suitable microbial hosts, e.g., Pseudomonas, themicrobes can be applied to the situs of the pest, where they willproliferate and be ingested by the pest. The result is a control of thepest. Alternatively, the microbe hosting the toxin gene can be treatedunder conditions that prolong the activity of the toxin and stabilizethe cell. The treated cell, which retains the toxic activity, then canbe applied to the environment of the target pest.

Where the tic900 toxin gene or a related nucleotide coding sequence isintroduced by means of a suitable vector into a microbial host, and thehost is applied to the environment in a living state, it is advantageousto use certain host microbes. For example, microorganism hosts can beselected which are known to occupy the pest's habitat. Microorganismhosts may also live symbiotically with a specific species of pest. Thesemicroorganisms are selected so as to be capable of successfullycompeting in the particular environment with the wild-typemicroorganisms, provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

A large number of microorganisms are known to inhabit the habitat ofpests. These microorganisms include bacteria, algae, and fungi. Ofparticular interest are microorganisms, such as bacteria, e.g., generaBacillus, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella,Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, e.g., genera Metarhizium, Bavaria, Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.

A wide variety of means are available for introducing a toxin geneencoding a toxin into a microorganism host under conditions that allowfor stable maintenance and expression of the gene. These methods arewell known to those skilled in the art and are described, for example,in U.S. Pat. No. 5,135,867.

As mentioned above, B. thuringiensis or recombinant cells expressing aTIC900 or related toxin can be treated to prolong the toxin activity andstabilize the cell. The pesticide microcapsule that is formed comprisesone or more TIC900 or related toxins within a cellular structure thathas been stabilized and will protect the toxin or toxins when themicrocapsule is applied to the environment of the target pest. Suitablehost cells may include either prokaryotes or eukaryotes, normally beinglimited to those cells that do not produce substances toxic to higherorganisms, such as mammals. However, organisms which produce substancestoxic to higher organisms could be used, where the toxic substances areunstable or the level of application sufficiently low as to avoid anypossibility of toxicity to a mammalian host. Of particular interest ashosts will be prokaryotes as well as lower eukaryotes such as fungi. Thecells of these organisms will usually be intact and be substantially inthe proliferative form when treated, rather than in a spore form,although in some instances spores may be employed. Such microcapsulescan also contain one or more TIC900 or related proteins along with oneor more unrelated insecticidal protein compositions including but notlimited to delta endotoxins insecticidal to lepidopteran species such asCry1, Cry2, and Cry9 proteins, as well as delta endotoxins insecticidalto coleopteran species such as Cry3, Cry22, ET70, ET80/76, ET33/34,PS149B1, ET100/101, and ET29 proteins and the like.

The cells generally will have enhanced structural stability that willenhance resistance to environmental conditions. Where the pesticide isin a proform or precursor form, the method of cell treatment should beselected so as not to inhibit processing of the proform to the matureform of the pesticide by the target pest pathogen. For example,formaldehyde will crosslink proteins and could inhibit processing of theproform of a polypeptide pesticide. The method of cell treatment retainsat least a substantial portion of the bio-availability or bioactivity ofthe toxin.

TIC900 and related coding sequences as set forth in SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19 and the like can beused as the basis for constructing modified nucleotide sequences forincorporation into plant cells. Even more preferable is the synthesis ofa non-naturally occurring nucleotide sequence that encodes a TIC900 orrelated insecticidal protein or its equivalent for expression in a plantcell, the synthesis of the non-naturally occurring nucleotide sequencebeing based on the amino acid sequence of the native protein withoutreference to the native nucleotide sequence from which the native aminoacid sequence was deduced. Expression of such sequences in plant cellswould render a plant comprised of such cells more resistant tolepidopteran species insect attack. Genetic engineering of plants withmodified sequences encoding one or more TIC900 or related proteins or arelated insecticidal amino acid sequence may be accomplished byintroducing the desired DNA containing the coding sequence into planttissues or cells, using DNA molecules of a variety of forms and originsthat are well known to those skilled in plant genetic engineering.Method for introducing nucleotide sequences into plants, plant cells andplant tissues are well known in the art.

DNA containing a modified gene encoding TIC900 or a related insecticidalprotein, operatively linked to a plant functional promoter, may bedelivered into the plant cells or tissues directly by a number of meansincluding but not limited to Agrobacterium mediated transformation,plant viruses, electroporation, microinjection, vacuum infiltration,liposome fusion means, and ballistic methods. The plant promoter may bea constitutive promoter; a temporally, spatially, chemically,photosynthetically, thermally, or artificially inducible promoter; atissue-specific promoter; or a chimeric or hybrid promoter assembledfrom parts of other plant functional promoters. For example, thepromoter may be a cauliflower mosaic virus (CaMV) 35S promoter or aplant functional derivative thereof.

Native bacterial genes and coding sequences are often poorly expressedin transgenic plant cells. Plant codon usage more closely resembles thatof other higher organisms than unicellular organisms, such as bacteria.Several reports have disclosed methods for improving expression ofrecombinant genes in plants (Murray et al., 1989, Nucleic AcidsResearch, Vol. 17:477-498; Diehn et al., 1998(b), Plant Physiology,117:1433-1443; Rocher et al., 1998, Plant Phys. 117:1445-1461). Thesereports disclose various methods for engineering coding sequences torepresent sequences which are more efficiently translated based on plantcodon frequency tables, improvements in codon third base position bias,using recombinant sequences which avoid suspect polyadenylation or A/Trich domains or intron splicing consensus sequences. While these methodsfor synthetic gene construction are notable, synthetic genes of thepresent invention for expression in particular plants are preparedsubstantially according to the method of Brown et al. (U.S. Pat. No.5,689,052).

The work described herein takes advantage of methods of potentiating inplanta expression of TIC900 and related insecticidal proteins, whichconfer resistance to lepidopteran insect pathogens, by incorporation orlocalization of coding sequences into the nuclear, plastid, orchloroplast genome of susceptible plants. U.S. Pat. No. 5,500,365 andrelated patents describe methods for synthesizing plant genes to achieveoptimum expression levels of the protein for which the synthesized,non-naturally occurring, synthetic, or artificial gene encodes. Thesemethods relate to the modification of native Bt structural genesequences to produce a coding sequence that is more “plant-like” andtherefore more likely to be translated and expressed by the plant,monocot or dicot. However, the method as disclosed in Brown et al. (U.S.Pat. No. 5,689,052) provides for enhanced expression of transgenes,preferably in monocotyledonous plants.

Thus, the amount of a gene coding for a polypeptide of interest, e.g. aTIC900 or related polypeptide, can be increased in plants bytransforming those plants using transformation methods mentioned above.In particular, chloroplast or plastid transformation can result indesired coding sequences being present in up to about 10,000 copies percell in tissues containing these subcellular organelle structures(McBride et al., WO 95/24492).

DNA encoding TIC900 and related proteins can also be introduced intoplants by utilizing a direct DNA transfer method into pollen asdescribed (Zhou et al., 1983, Mol. Cell. Biol., 10:4529-4537; Hess,1987, Hess, Intern Rev. Cytol., 107:367.). Expression of polypeptidecoding sequences, i.e., tic900 and the like, can be obtained byinjection of the DNA into reproductive organs of a plant as described(Pena et al., 1987, Nature, 325:274). The DNA can also be injecteddirectly into the cells of immature embryos and into rehydrateddesiccated embryos as described (Neuhaus et al., 1987, Theor. Appl.Genet., 75:30).

After effecting delivery of exogenous nucleotide sequences encodingTIC900 or related proteins to recipient cells, the next step to obtain atransgenic plant generally concerns identifying the transformed cellsfor further culturing and plant regeneration, i.e., selection of thetransformed cells. As mentioned herein, in order to improve the abilityto identify transformants, one may desire to employ a selectable orscreenable marker gene as, or in addition to, the expressible gene ofinterest. In this case, one would then generally assay the potentiallytransformed cell population by exposing the cells to a selective agentor agents, or one would screen the cells for the desired marker genetrait.

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the transformed cultures to a selective agent, such asa metabolic inhibitor, an antibiotic, herbicide or the like. Cells thathave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.One example of a preferred marker gene confers resistance to theherbicide glyphosate. When this gene is used as a selectable marker, theputatively transformed cell culture is treated with glyphosate. Uponexposure to glyphosate, transgenic cells containing a recombinant GOXenzyme or a recombinant glyphosate insensitive EPSPS enzyme will beavailable for further culturing while sensitive, or non-transformedcells, will not. (U.S. Pat. No. 5,569,834). Another example of apreferred selectable marker system is the neomycin phosphotransferase(nptII) resistance system by which resistance to the antibiotickanamycin is conferred, as described in U.S. Pat. No. 5,569,834. Again,after transformation with this system, transformed cells will beavailable for further culturing upon treatment with kanamycin, whilenon-transformed cells will not. Yet another preferred selectable markersystem involves the use of a gene construct conferring resistance toparomomycin. Use of this type of a selectable marker system is describedin U.S. Pat. No. 5,424,412. Other selectable markers are well known inthe art, including but not limited to antibiotic resistance markers suchat nptII, tet, aad, and the like, phnO and other various acetylases(U.S. Pat. No. 6,448,476), various esterases (U.S. Pat. No. 6,107,549),barnase (Hartley, 1988), J. Mol. Biol. 202: 913), bacterial enzymesconferring glyphosate oxidase activity upon the transformed cell (gox)(Barry et al., 1992, Inhibitors of amino acid biosynthesis: Strategiesfor imparting glyphosate tolerance to crop plants. In: Biosynthesis andMolecular Regulation of Amino Acids in Plants. pp. 139-145. Singh,Flores, and Shannon Eds., American Society of Plant Physiologists,Rockville, Md.) and the like.

Transplastonomic selection (selection of plastid or chloroplasttransformation events) is simplified by taking advantage of thesensitivity of chloroplasts or plastids to spectinomycin, an inhibitorof plastid or chloroplast protein synthesis, but not of proteinsynthesis by the nuclear genome encoded cytoplasmic ribosomes.Spectinomycin prevents the accumulation of chloroplast proteins requiredfor photosynthesis so spectinomycin resistant transformed plant cellsmay be distinguished on the basis of their difference in color: theresistant, transformed cells are green, whereas the sensitive cells arewhite, due to inhibition of plastid-protein synthesis. Transformation ofchloroplasts or plastids with a suitable bacterial aad gene, or with agene encoding a spectinomycin resistant plastid or chloroplastfunctional ribosomal RNA provides a means for selection and maintenanceof transplastonomic events (Maliga, 1993, Trends in Biotechnology11:101-106).

It is further contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such asglyphosate or kanamycin, may either not provide enough killing activityto clearly recognize transformed cells or may cause substantialnonselective inhibition of transformants and non-transformants alike,thus causing the selection technique to not be effective. It is proposedthat selection with a growth inhibiting compound, such as glyphosate orAMPA (amino-methyl phosphoric acid) at concentrations below those thatcause 100% inhibition, followed by screening of growing tissue forexpression of a screenable marker gene such as kanamycin would allow oneto recover transformants from cell or tissue types that are not amenableto selection alone. It is proposed that combinations of selection andscreening may enable one to identify transformants in a wider variety ofcell and tissue types.

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art. Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

The development or regeneration of plants containing a foreign,exogenous gene that encodes a TIC900 or related polypeptide introducedinto the plant genome by Agrobacterium transformation of leaf explantscan be achieved by methods well known in the art (Horsch et al., Science227:1229-1231; 1985). In this procedure, transformants are cultured inthe presence of a selection agent and in a medium that induces theregeneration of shoots in the plant strain being transformed asdescribed (Fraley et al., PNAS, USA 80:4803; 1983). In particular, U.S.Pat. No. 5,349,124 details the creation of genetically transformedlettuce cells and plants resulting therefrom which express hybridcrystal proteins conferring insecticidal activity against Lepidopteranlarvae to such plants.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, or pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important,preferably inbred lines. Conversely, pollen from plants of thoseimportant lines is used to pollinate regenerated plants. A transgenicplant of the present invention containing a nucleotide sequence encodinga desired TIC900 or related polypeptide is cultivated using methods wellknown to one skilled in the art.

A transgenic plant of this invention thus has an increased amount of acoding region encoding a TIC900 or related polypeptide. A preferredtransgenic plant is an independent segregant and can transmit that geneand its activity to its progeny. A more preferred transgenic plant ishomozygous for that gene, and transmits that gene to all of itsoffspring on sexual mating. Seed from a transgenic plant may be grown inthe field or greenhouse, and resulting sexually mature transgenic plantsare self-pollinated to generate true breeding plants. The progeny fromthese plants become true breeding lines that are evaluated for increasedexpression of the B. thuringiensis transgene. To identify a transgenicplant expressing high levels of a TIC900 or related protein from apreferred nucleotide sequence, it is necessary to screen the selectedtransgenic event, (Ro generation) for insecticidal activity and/orexpression of the gene. This can be accomplished by various methods wellknown to those skilled in the art, including but not limited to: 1)obtaining small tissue samples from the transgenic Ro plant and directlyassaying the tissue for activity against susceptible insects, e.g.,European corn borer (ECB), tobacco budworm (TBW) and diamondback moth(DBM), in parallel with tissue derived from a non-expressing, negativecontrol plant; 2) analysis of protein extracts by enzyme linkedimmunoassays (ELISA) specific for the TIC900 or related protein; or 3)reverse transcriptase thermal amplification (also known in the art asrtPCR) to identify events expressing the sequence encoding the TIC900 orrelated protein.

The following examples further illustrate the characteristics of thenucleotide sequences disclosed herein and the insecticidal activity ofthe proteins encoded by the disclosed nucleotide sequences. In addition,methods and procedures for practicing the invention are disclosed.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Examples Example 1 Preparation and Bioassay of B. Thuringiensis StrainEG5438 Culture Supernatant

B. thuringiensis strain EG5438 was grown in 60 ml of PYG culture mediumwith shaking overnight at 30° C. PYG medium contained the following:11.8 g peptone, 23.6 g yeast extract, 4 ml glycerol, 19.4 g K₂HPO₄anhydrous, and 2.2 g KH₂PO₄ anhydrous. Deionized water was added to 1liter, and the medium was autoclaved for 15 min The B. thuringiensisculture was centrifuged at 11,000×g for 30 min and the supernatant wastransferred to a clean flask. The supernatant was chilled to 4° C., and34 grams of ammonium sulfate plus 1 ml of 1 M NaOH were slowly added tothe supernatant while stirring. The mixture was centrifuged and theresulting pellet was dissolved in 2 ml of 20 mM Tris-HCl pH 7.5. Thesolution was transferred to dialysis tubing (6000 MWCO) and was dialyzedat 4° C. against 20 mM Tris-HCl pH 7.5. This is referred to as thedialyzed supernatant.

The dialyzed supernatant was tested for toxicity to diamondback moth(DBM) larvae as follows. Fifty μl of the dialyzed supernatant wasapplied topically to 2 ml of insect diet in a cup. A total of thirty-twodiet cups were treated with the dialyzed supernatant. As a controlsixty-four diet cups were not treated with dialyzed supernatant. Onefirst-instar DBM larva was placed in each diet cup and insect mortalitywas scored after 7 days. For larvae on the untreated control diets 1larvae out of 64 (2%) died. For larvae on the diets treated with thedialyzed supernatant 29 out of 32 (90%) died, suggesting that thedialyzed supernatant of strain EG5438 contained one or more factorstoxic to DBM larvae.

Example 2 Fractionation of Proteins in the Dialyzed Supernatant andBioassay of Protein Fractions

Proteins in the dialyzed supernatant were initially fractionated bysodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Thirty μl of the dialyzed supernatant was mixed with 15 μl of proteinsolubilization buffer, the mixture was heated to 100° C. for 5 min, and25 μl of the mixture was applied to a polyacrylamide gel. An electriccurrent was applied to the gel to size-separate proteins into the gel.The proteins were visualized after electrophoresis by staining withCoomassie dye. The dialyzed supernatant contained approximately twentyproteins ranging in size from approximately 20 kDa to about 100 kDa.

Proteins in the dialyzed supernatant were fractionated by DEAEion-exchange chromatography. Two ml of the dialyzed supernatant wasapplied to a 1 ml DEAE column The column was washed with 10 ml of 20 mMTris-HCl, pH 7.5, followed by washing with 20 ml of a 0 to 1 M NaClgradient in 20 mM Tris-HCl, pH 7.5. Fractions of 1 ml were collected.Each fraction was dialyzed against 20 mM Tris-HCl, pH 7.5, andindividual fractions were tested for toxicity to DBM larvae as describedabove. Fractions with the highest toxicity were collected and combinedand referred to as the DEAE pool.

The DEAE pool was applied to a carboxymethyl cellulose (CM) ion exchangecolumn The column was washed with 10 ml of 20 mM Tris-HCl, pH 7.5,followed by washing with 20 ml of a 0 to 1 M NaCl gradient in 20 mMTris-HCl, pH 7.5. One ml fractions were collected and dialyzed against20 mM Tris-HCl, pH 7.5. These fractions were referred to as the CMfractions. The CM fractions were tested for toxicity to DBM larvae. Thisanalysis showed that CM fractions had the highest toxicity to DBM andcontained a protein of approximately 66 kDa. The 66 kDa protein wasreferred to as the 5438-66 protein, also referred to as secreted TIC900,TIC900s, or mTIC900 (referring to a mature form of the proteinidentified in the culture supernatant that may be different from anyprecursor TIC900 protein (pTIC900) not yet released from the cell).

Example 3 Determination of the N-Terminal Sequence of a Fragment of aTIC900 Protein

mTIC900 protein was purified from the supernatant of strain EG5438 byDEAE and CM ion exchange chromatography. Attempts to determine theN-terminal sequence of the purified mTIC900 protein by standard methodswere not successful. To overcome this difficulty, mTIC900 protein wasfragmented by cyanogen bromide treatment (Cordoba et al., J. Biochem.Biophys. Methods 35: 1, 1997). The cyanogen bromide-generated TIC900fragments were size-separated by SDS-PAGE without Coomassie stainingSeparated TIC900 fragments were transferred from the SDS-PAGE to apolyvinylidene difluoride (PVDF) membrane by an electro transfer. ThePVDF membrane was stained briefly with Coomassie dye and a portion ofthe membrane containing an approximately 14 kDa fragment of TIC900protein was excised with a razor blade. The excised PVDF membranecontaining the 14 kDa fragment was subjected to automated Edmundsequencing, revealing the amino acid sequence as shown in SEQ ID NO:1,in which the Xaa amino acid residue at position one (1) wasindeterminable except that it was presumed to be either a Serine,Tyrosine, Aspartate or Histidine residue, but most likely a Tyrosineresidue, Xaa amino acid residue at position 15 was indeterminable exceptthat it was likely a Proline residue, and the Xaa residue at position 18was also indeterminable except that it was likely an Arginine residue.

Example 4 Cloning tic900 Gene Encoding TIC900 Protein

Based on the sequence obtained from the 14 kDa TIC900 protein fragment(SEQ ID NO:1), a gene-specific oligonucleotide was designed. Due to thedegeneracy of the genetic code it is not possible to know the exactsequence of a gene based on the sequence of the protein encoded by thegene. Therefore for amino acids that can be encoded by more than asingle codon, it is necessary to guess at the correct codon. The chanceof guessing accurately is improved by the fact that the B. thuringiensisgenome is approximately 68% AT (adenosine and thymidine). Therefore, foramino acids encoded by more than one codon, the codon or codons whichcontain A's and T's are selected, and for codons that containsubstantially G/C, those codons that have a degeneracy in the third baseposition are selected preferentially based on whether the third base isan A or a T nucleotide. An oligonucleotide designated WD470 (SEQ IDNO:2) was designed which is one of many that could conceivably encodethe amino acid set forth in SEQ ID NO:1, taking into consideration theA/T usage in Bacillus thuringiensis for codons encoding any given aminoacid.

DNA was purified from B. thuringiensis strain EG5438 cells by standardprocedures. Samples of the EG5438 DNA were subjected to either HindIIIor EcoRI restriction enzyme digestion and were size-fractionated byelectrophoresis through an agarose gel and subjected to Southern blotanalysis using an alkaline phosphatase conjugated WD470 oligonucleotideprobe. After incubation for approximately 16 hours at 40° C. the blotwas washed, treated with chemiluminescent buffer, and exposed to x-rayfilm. The WD470 probe specifically hybridized with EG5438 DNArestriction fragments that were approximately 2.5 kb (HindIII) and 3.0kb (EcoRI) in length, respectively.

A library of EG5438 DNA consisting of about 3.0 kb EcoRI fragments wasconstructed in a CIP (calf intestine phosphatase) treated EcoRI digestedpUC18 plasmid. The library was transformed by electroporation into an E.coli XL1BLUE strain and plated to LB-ampicillin. Colonies that arosewere blotted to a membrane and probed with the alkaline phosphataseconjugated WD470 oligonucleotide probe. Several positive clones wereselected and plasmid DNA was obtained from each. Plasmid DNA's weredigested with EcoRI to confirm the presence of a single EcoRI insertconsisting of about 3.0 kb. Plasmids were also subjected tohybridization to the alk-phos conjugated WD470 probe to confirm thecomplementarity of the probe and inserted DNA. A single clone wasselected for further analysis and was designated as plasmid pEG1398. Theinserted DNA in pEG1398 was subjected to sequence analysis. A sequencecontaining a partial open reading frame consisting of nucleotideposition 1176 through 1803 as set forth in SEQ ID NO:3 was obtained, aswell as an additional 24 nucleotides beyond nucleotide 1803 (data notshown) which contained a termination codon immediately after nucleotidesat position 1801-1803 as set forth in SEQ ID NO:3.

The complete sequence of an ORF encoding the TIC900 protein was notpresent within the EcoRI fragment cloned into plasmid pEG1398.Oligonucleotide primers specific for the 5′ and 3′ ends of the sequenceidentified therein were designed to enable the synthesis of a labeledprobe for use in detecting a larger cloned fragment of EG5438 DNA thatlikely contained the full length ORF encoding the TIC900 protein. Adigoxygenin labeled DNA probe was prepared by amplification using theprimers and the inserted DNA in pEG1398 as a template. The DIG-labeledDNA was used to probe a Southern blot of EG5438 DNA that had beenresolved in an agarose gel after digestion with various restrictionenzymes. A HindIII fragment about 2.5 kb in length was identified as afragment that could contain the full length ORF encoding the TIC900protein.

A EG5438 DNA fragment of about 2.5 kb was cloned using a means similarto that described above for the about 3.0 kb EcoRI fragment except thatthe HindIII fragment was cloned into a pBlueScript KS plasmid and theprobe used was a DIG-labeled DNA segment consisting of a part of theopen reading frame identified within the 3.0 Kb EcoRI fragment in theplasmid pEG1398. One plasmid containing an approximately 2.5 kb HindIIIfragment that hybridized to the DIG-labeled EcoRI fragment presentwithin pEG1398 was selected for further analysis and designated asplasmid p5438-2.5-kb-H3. The recombinant E. coli strain harboringp5438-2.5-H3 was designated as 5438 2.5 kb H3. The DNA sequence of the2.5 kb HindIII insert in the plasmid p5438-2.5-kb-H3 was determined, andtranslation of this sequence in all six reading frames revealed an openreading frame of 1803 nucleotides, the sequence of which is set forth inSEQ ID NO:3.

The ORF from nucleotide position 1 through nucleotide position 1803 asset forth in SEQ ID NO:3 is predicted to encode a protein of about68,868 Daltons, which has been designated herein as TIC900. The aminoacid sequence of the predicted precursor form of a TIC900 protein(pTIC900) deduced from the open reading frame in SEQ ID NO:3 is shown asset forth in SEQ ID NO:4. Identity and similarity comparison of theamino acid sequence of the deduced TIC900 amino acid sequence (SEQ IDNO:4) with the GenBank protein database revealed that the nearestidentity was to a Cry1 Ca protein exhibiting about 49% identity.

Example 5 Expression of a Cloned tic900 Gene in Recombinant B.Thuringiensis

B. thuringiensis insecticidal toxin genes are often poorly expressed inrecombinant E. coli strains. B. thuringiensis strain EG10650 is anacrystalliferous strain that was designed for use as a recipient strainfor testing whether cloned Bt genes encode insecticidal proteins.(EG10650, NRRL Accession Number NRRL B-30217, U.S. Pat. No. 6,468,52).The TIC900 coding sequence on the cloned HindIII fragment in plasmidp5438-2.5 kb-H3 was transferred into the HindIII restriction site in theB. thuringiensis-E. coli shuttle vector pEG597 (Baum, J. A.; Coyle, D.M.; Gilbert, M. P.; Jany, C. S.; Gawron-Burke, C., 1990 Novel cloningvectors for Bacillus thuringiensis; Applied and EnvironmentalMicrobiology 56 (11): 3420-3428) resulting in the construction ofplasmid pMON74010 which confers chloramphenicol resistance to recipientBacillus cells. Plasmid pMON74010 was transformed by electroporationinto the acrystalliferous B. thuringiensis strain EG10650 yieldingstrain SIC9002. Strain EG10650 was grown as a control in PYG medium asdescribed in Example 1. The recombinant strain SIC9002 was grown in PYGmedium plus 5 ug/ml chloramphenicol. Culture supernatants were preparedas described in Example 1. Proteins in the culture supernatants wereresolved by standard SDS-PAGE analysis and were visualized afterstaining with Coomassie brilliant blue. The SDS-PAGE analysis resultsrevealed that strains EG10650 and SIC9002 secreted similar numbers andsizes of proteins into their respective culture supernatants with theexception that the culture supernatant of strain SIC9002 contained aprotein of approximately 66 kDa which did not appear to be present inthe culture supernatant of strain EG10650. This result suggested thatthe cloned tic900 open reading frame in p5438-2.5 kb-H3 encoded aprotein that migrated with a mass of approximately 66 kDa in SDS-PAGEgels. A discrepancy in the size of the amino acid sequence deduced fromthe ORF as set forth in SEQ ID NO:3 (about 69 kDa) and the observed massby migration in SDS-PAGE suggests that the secreted form of the proteinmay in fact be reduced in size by about 2500 to 3000 Da. This is notunexpected since most secreted proteins exhibit some proteolyticreduction in size as they are passed through any secretion machinery.However, there is no apparent type II signal peptide present as judgedfrom an analysis of the primary amino acid sequence of the precursorTIC900 protein (pTIC900).

Example 6 Bioassay of TIC900 Protein Produced from the Cloned tic900Coding Sequence

Culture supernatants of strains EG10650 and SIC9002 were applied to thesurface of insect diet as described herein above. First instar Europeancorn borer (ECB) larvae and tobacco budworm (TBW) eggs were placed ontreated diet and were allowed to develop for 1 week. Insect larvae werevisually evaluated. ECB larvae and TBW larvae reared on untreated dietor on diet treated with EG10650 supernatant exhibited normal growth. Incontrast, ECB larvae and TBW larvae reared on diet treated with SIC9002supernatant exhibited significant stunting. These results suggested thatthe protein produced from expression of the cloned tic900 gene inhibitedgrowth of ECB and TBW larvae.

Example 7 Identification of Strains Containing tic900 Homologs

A DIG-labeled probe encompassing the entire open reading frame of thetic900 coding sequence was prepared using the following thermalamplification primers:

5′-gcgctagcatgaattcaaaggaacatgattatctaaaag-3′, SEQ ID NO: 21, and5′-cgggctcgagctattcaacaggaataaattcaattttatcc-3′, SEQ ID NO: 22.

Between one and five μg genomic DNA from a collection of Bt strains wasdigested to completion with HindIII and the resulting fragments wereresolved as a smear on an agarose gel. The gel was used in a Southernblot procedure in which the resolved DNA was denatured, transferred to anylon membrane, fixed, and exposed to the DIG labeled probe describedabove. Hybridization was carried out in DIG Easy Hybe (Roche) at 42° C.(DIG Easy Hybe at 42° C. is equivalent to a stringent 42° C.hybridization with a hybridization buffer system containing 50%formamide). Moderately stringent washes were performed as follows: 1)one time for 5 minutes and one time for 15 minutes at 25° C. in 2×SSC,0.1% SDS; and 2) two times for 15 minutes each at 65° C. in 0.5×SSC,0.1% SDS.

Thirteen strains were identified that contained from between one andthree HindIII fragments that hybridized to the tic900 probe. DNA fromeach of these strains was used as a template for thermal amplificationof tic900 homologs. Primers set forth as SEQ ID NO:21 and SEQ ID NO:22were used to amplify tic900 homologs using the Expand High Fidelity PCRkit (Roche). Thermal amplification reaction conditions consisted of a 50μL volume comprising 200 μM each dNTP, 300 nM each primer, 0.1-250 nggenomic DNA template, and 2.6 units enzyme mix in 1× reaction buffer(supplied by the manufacturer with the reagents in 10× concentrate).

Thermal amplification cycles consisted of one cycle of 2 minutes at 94°C.; ten cycles of 15 seconds at 94° C., 30 seconds at 60° C., and 2minutes at 72° C.; followed by twenty five cycles of 15 seconds at 94°C., 30 seconds at 60° C., and 2 minutes at 72° C., increasing each ofthe last twenty five cycles by 5 seconds per cycle; and a terminalextension phase of 7 minutes at 72° C. at the end of the last cycle.

DNA from nine of the thirteen strains subjected to this thermalamplification reaction produced amplification products (amplicons) thatwere subsequently cloned and sequenced. The 5′ and 3′ end sequences ofthe cloned thermal amplification products were fixed by the sequences ofthe primers and may not be representative of the sequence of the nativegene throughout the sequence established by the amplification primers.Regardless, the amplicon sequences were substantially the same as thefull length native sequences expressed for analysis of insecticidalactivity. One skilled in the art will realize that the amplicons can beused as probes to fish out the full-length native sequences encodinginsecticidal proteins related to the TIC900 protein. The proteinsencoded by the open reading frame for each thermal amplification productand the strains from which each thermal amplification product wereobtained are indicated in Table 1, as shown above.

Variant amplification primers and multiple amplification conditions werealso used to identify tic900 homologs from the CRW-active Bt strainEG3907. The tic900 homolog in EG3907 was mapped by southern blot tofacilitate cloning of the open reading frame encoding this protein. Thetic900 homolog in EG3907 had a different HindIII restriction patternthan that of the tic900 gene from EG5438. The EG3907 homolog wasidentified on an approximately 13 kb BamHI/BglII fragment.BamHI/BglII-digested EG3907 DNA was ligated into BamHI-digested phagelambda GEM-11 arms. Southern blots of DNA from an additional 30 Btstrains exhibiting CRW activity in the fermentation broth identified 2strains, EG3291, EG3388, containing DNA that hybridized to a tic900probe under stringent conditions. Both of these strains exhibitedidentical HindIII restriction patterns, but these were different fromthe restriction pattern containing the tic900 sequence from strainEG5438 as set forth in SEQ ID NO:3, and different from the restrictionpattern containing the homolog identified as being present in strainEG3907.

DNA (0.5 μg) from 132 Bt strains was dot-blotted to Nytran membranes andprobed with a tic900 DNA probe under stringent conditions. DNA from thefifteen strains exhibiting the strongest tic900 hybridization signalswere analyzed further. DNA from each strain was digested to completionwith HindIII and subjected to a Southern blot procedure as describedabove. DNA from several strains that appeared to hybridize in the dotblots did not exhibit strong hybridization signals using the Southernblot method. 14 strains containing sequences homologous to the tic900gene have been analyzed using HindIII Southern blots. Based on thehybridization profiles that appear using HindIII digestion, at least 4different tic900 homologs are present in these strains.

The following Bacillus thuringiensis strains exhibit HindIII fragmentsthat hybridize to a tic900 probe under stringent or specifichybridization conditions: EG3291, EG3388, EG3879, EG3907, EG4090,EG4092, EG4293, EG4332, EG4577, EG4611, EG4963, EG4971, EG5023, EG5438,and EG5526. These strains also produce extracellular proteins that canbe evaluated for insecticidal activity. Depending on the strainselected, the hybridizing HindIII fragments varied in size from about0.8 kb to about 6.3 kb. The nucleotide sequence of each fragment thathybridized to the tic900 probe was determined, and open reading frameswere deduced from these sequences, each set forth herein as SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, and SEQ ID NO:19. The amino acid sequence of aprotein comparable in size to that of TIC900 was deduced from each ofthese open reading frames, as set forth respectively in SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:30, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, and SEQ ID NO:20. These deduced amino acidsequences were designated respectively as TIC402, TIC403, TIC404,TIC434, TIC961, TIC962, TIC963, TIC965, and TIC966. As set forth inTable 1, these nucleotide sequences were sourced respectively from thefollowing B. thuringiensis strains: EG3879 (TIC402), EG4332 (TIC403),EG4971 (TIC404), EG4090 (TIC961), EG4293 (TIC962), EG4611 (TIC963 andTIC434), EG5023 (TIC965), and EG4092 (TIC966). An additional strain wasidentified that exhibited a sequence that hybridized to the tic900probe. Strain EG5526 contained a HindIII fragment that was apparentlyidentical in size to the HindIII fragment identified as encoding TIC402from strain EG3879. DNA sequence analysis revealed that the EG5526fragment contained an ORF (TIC964) that was identical in sequence tothat of the tic402 ORF. Acrystalliferous strains of B. thuringiensiscontaining plasmids encoding these cloned homologs of tic900 were eachsubjected to insect bioassay and were determined to exhibit insecticidalbioactivity.

There is a high degree of identity between the sequences encoding theproteins of the present invention. In fact, an alignment of the openreading frames including each of the tic genes reveals that none of theORF's are less than about 97% identical to each other. An alignment ofthe amino acid sequences encoded by each of the ORF's also indicatesthat there is a high degree of identity between the proteins of thepresent invention. TIC962 and TIC963 are the most distantly related, butstill very closely related in that they exhibit greater than a 96%identity at the amino acid sequence level. Most changes to thenucleotide sequence for any given change in any ORF in relation to aconsensus sequence established based on an alignment of all of thenucleotide sequences indicates that the changes are silent in that theyaffect only the third base in a codon and result most often in nomodification of the encoded amino acid sequence.

Subcultures of B. thuringiensis strains EG5438 containing the nativetic900 gene, and SIC9002 containing the cloned tic900 coding sequencewere deposited in the permanent collection of the Agricultural ResearchService Culture Collection, Northern Regional Research Laboratory(NRRL), U.S. Department of Agriculture (USDA), 1815 North UniversityStreet, Peoria, Ill. 61604, USA. B. thuringiensis strain SIC9002 wasdeposited on Apr. 25, 2002 and provided with the NRRL accession numberNRRL B-30582. B. thuringiensis strain EG5438 was deposited on May 3,2002 and was provided with the NRRL accession number NRRL B-30584.

Example 8 Genes Encoding Chimeric Insecticidal Proteins

This example illustrates that the TIC900 class of proteins exhibitsimilarities with the Cry1 class of Bt insecticidal proteins and that achimeric protein can be constructed from all or a part of a TIC900 classprotein linked in frame with all or a part of a Cry1 protein and testedfor insecticidal activity.

Comparison of any of the TIC900 class of proteins disclosed herein withother Bt insecticidal proteins suggests that these proteins are mostclosely related to the Cry1 classes of proteins, and in particular tothe insecticidal portion of the Cry1 proteins. The TIC900 class ofproteins exhibit structural similarities to the Cry1 protein toxinportions in that the Cry1 proteins exhibit a domain structure consistingof a first domain consisting of about the first 200 to about the first240 amino terminal amino acids which is referred to as domain I, asecond domain that consists of about amino acids 240 through about aminoacid 400 or so which is referred to as domain II, and a carboxy-terminaldomain referred to as domain III consisting of amino acids from aboutresidue 400 or so through the end of the toxin domain. The TIC900 classof proteins appear to exhibit this type of domain structure even thoughthe TIC900 class of proteins generally are not as long as most Cry1toxin domains. It has previously been shown that Cry1 toxin domains canbe fused to heterologous protoxin peptide structures, and that thefusions result in crystal formation, and often also retain insecticidalbioactivity when the resulting crystals are tested in bioassay. A fusionprotein (SEQ ID NO:24, TIC109) was constructed in which TIC900 was fusedto the Cry1 Ac protoxin peptide structure. The fusion protein wasexpressed from the nucleotide sequence as set forth in SEQ ID NO:23 inpMON74119 in B. thuringiensis strain EG10650 (recombinant straindesignated as SIC1047). SEQ ID NO:23 corresponds to a TIC900 codingsequence from nucleotide position 1-1809, and a Cry1Ac protoxin domaincoding sequence from nucleotide position 1816-3504. The chimeric proteinTIC109 formed in SIC1047 fermentations produced crystalline inclusions,which were tested in bioassay against Tobacco Budworm, Corn Earworm, andFall Armyworm. The chimeric protein exhibited bioactivity similar tothat exhibited by TIC900, but was not biologically active against FallArmyworm.

TIC110 (SEQ ID NO:26) encoded by the nucleotide sequence as set forth inSEQ ID NO:25 is a Cry1F/TIC900 chimeric insecticidal protein linked to aCry1Ac protoxin peptide sequence. SEQ ID NO:25 corresponds to a sequenceencoding Cry1F domain I from about nucleotide position 1-723, a sequenceencoding TIC900 domains II and III from about nucleotide position724-1809, and a Cry1 Ac coding sequence from about nucleotide position1810-3510. This protein can be expressed in an acrystalliferous strainof Bt and the crystalline protein inclusions tested in bioassay todetermine the biological activity against various lepidopteran pestspecies.

TIC111 (SEQ ID NO:28) is encoded by the nucleotide sequence as set forthin SEQ ID NO:27. TIC111 corresponds to an insecticidal chimeric proteinconsisting of a Cry1Ac domain I linked to TIC900 domains II and III,which is linked to a Cry1Ac protoxin domain. TIC111 can be expressedfrom pMON74122 and the crystalline protein inclusions tested in bioassayto for bioactivity against various lepidopteran pest species.

pMON74122 was transformed into the acrystalliferous Bt strain EG10650resulting in the transformed host cell SIC1049 expressing the TIC111protein. TIC111 crystals were collected and tested in bioassay againstblack cutworm (BCW), Diamondback Moth (DBM), Tobacco Budworm (TBW), CornEarworm (CEW), and Fall Armyworm (FAW). Insecticidal bioactivity wasobserved for BCW, DBM and TBW, consistent with the insecticidalbioactivity for TIC900.

In summary, the above detailed description describes the presentinvention. It will be understood by those skilled in the art that,without departing from the scope and spirit of the present invention andwithout undue experimentation, the present invention can be performedwithin a wide range of equivalent parameters. While the presentinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications. The present invention is intended to include any uses,variations, or adaptations of the invention following the principles ofthe invention in general. Various permutations and combination of theelements provided in all the claims that follow are possible and fallwithin the scope of this invention.

Reference to the word ‘comprising’ or ‘comprise’ or ‘comprises’ whetherin the claim language or in the specification is intended to be definedas a term or terms meaning “includes at least”.

All publications and patents mentioned in this specification are hereinincorporated by reference as if each individual publication or patentwas specially and individually stated to be incorporated by reference.

REFERENCES

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1. A recombinant polynucleotide which encodes a Bacillus thuringiensis insecticidal toxin protein or insecticidal fragment thereof, active against a lepidopteran insect pest, wherein said insecticidal toxin protein comprises a polypeptide sequence that has at least about 80% sequence identity to SEQ ID NO:6 (TIC402).
 2. The recombinant polynucleotide of claim 1, wherein said insecticidal toxin protein comprises SEQ ID NO:6.
 3. The recombinant polynucleotide of claim 1, wherein said lepidopteran insect pest is selected from the group consisting of a Noctuidae, a Tortricidae, Epinotia aporema, Anticarsia gemmatalis, Pseudoplusia includens, European Corn Borer (ECB), a Tobacco Budworm (TBW), Black Cutworm (BCW), and a Diamondback Moth (DBM).
 4. The recombinant polynucleotide of claim 1, wherein said toxin has a molecular weight between approximately 65 kDa and approximately 70 kDa, and wherein said insecticidal toxin is SEQ ID NO:6 (TIC402).
 5. (canceled)
 6. The recombinant polynucleotide of claim 1, wherein said nucleotide sequence has been optimized for expression in plants.
 7. The recombinant polynucleotide of claim 6, wherein said nucleotide sequence has been optimized for (a) expression in a monocot plant, said optimization comprising one or more of the steps selected from the group consisting of (i) removing polyadenylation sequences, (ii) adjusting the A and T content of the nucleotide sequence to be from about 40% to about 49% without modifying the amino acid sequence of the protein, and (iii) modifying codons in the coding sequence to be consistent with the steps (i) and (ii), or (b) expression in a dicot plant, said optimization comprising one or more of the steps selected from the group consisting of (i) removing polyadenylation sequences, (ii) adjusting the A and T content of the nucleotide sequence to be from about 40% to about 49% without modifying the amino acid sequence of the protein, and (iii) modifying codons in the coding sequence to be consistent with the steps (i) and (ii).
 8. An insecticidal protein active against Lepidopteran insects, said protein comprising an amino acid sequence that has at least about 80% sequence identity to SEQ ID NO:6 (TIC402).
 9. (canceled)
 10. A host cell comprising the recombinant polynucleotide of claim
 1. 11. The host cell of claim 10, wherein said host cell is a plant cell.
 12. A method for controlling a lepidopteran insect pest, said method comprising contacting said pest with a pesticidal amount of the insecticidal protein of claim 8 or insecticidal fragment thereof. 13-14. (canceled)
 15. The host cell of claim 11, said plant cell selected from a corn plant cell, a wheat plant cell, a rice plant cell, an oat plant cell, an onion plant cell, and a grass plant cell; and wherein said dicot plant cell comprises a cotton plant cell, a canola plant cell, a soybean plant cell, a tobacco plant cell, a fruit tree plant cell, a cruciferous plant cell, a pepper plant cell, an ornamental plant cell, a sunflower plant cell, a cucurbit plant cell, and a melon plant cell.
 16. A method for expressing a lepidopteran-active toxin protein in a plant, comprising the steps of: (a) inserting into the genome of a plant cell a nucleic acid sequence comprising in the 5′ to 3′ direction an operably linked recombinant, double-stranded DNA molecule, wherein the recombinant, double-stranded DNA molecule comprises: (i) a promoter that functions in the plant cell; (ii) a nucleotide sequence encoding an insecticidal amino acid sequence having at least about 80% sequence identity to SEQ ID NO:6; and (iii) a 3′ non-translated nucleotide sequence that functions in the cells of the plant to cause termination of transcription; (b) obtaining a transformed plant cell containing the nucleic acid sequence of step (a); and (c) generating from said transformed plant cell a plant that expresses the lepidopteran-active toxin protein in the transformed plant. 17-20. (canceled)
 21. A plasmid vector comprising the recombinant polynucleotide of claim
 1. 22. A transformed plant comprising the recombinant polynucleotide of claim
 1. 23. A seed from the transformed plant of claim 22, wherein said seed comprises said recombinant polynucleotide.
 24. A biological sample derived from the tissues or seed of the plant of claim 22, said sample comprising a detectable amount of said recombinant polynucleotide.
 25. A commodity product derived from the plant of claim 22, wherein said product comprises a detectable amount of said recombinant polynucleotide.
 26. A composition comprising an insecticidally effective amount of the insecticidal protein of claim
 8. 27. The composition of claim 26, said composition further comprising an additional insecticidal agent toxic to the same Lepidopteran insect pest but exhibiting a different mode of effectuating its insecticidal activity from said insecticidal protein.
 28. The composition of claim 27, wherein said insecticidal agent is selected from the group consisting of a Bacillus toxin, a Xenorhabdus toxin, a Photorhabdus toxin, and a dsRNA specific for suppression of one or more essential genes in said insect pest.
 29. The composition of claim 28, wherein said Bacillus toxin is selected from the group of proteins consisting of a Cry1, a Cry2, and a Cry9 toxin.
 30. A method of controlling Lepidopteran insect infestation in a crop plant and providing insect resistance management, said method comprising contacting said crop plant with a pesticidal amount of the composition of claim
 26. 